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. 1992 Feb;174(4):1268–1272. doi: 10.1128/jb.174.4.1268-1272.1992

DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

J M Boyd 1, K C Hall 1, J R Murphy 1
PMCID: PMC206420  PMID: 1735717

Abstract

The structural gene encoding DtxR, an iron-dependent diphtheria tox regulatory element, has recently been cloned and sequenced from the C7(-) strain of Corynebacterium diphtheriae (J. M. Boyd, M. Oza, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 87:5972, 1990). We report here the molecular cloning, DNA sequence analysis, and characterization of DtxR from the PW8(-), 1030(-), and C7hm723 strains of C. diphtheriae. While the sequence of dtxR from PW8(-) is identical to that of the C7(-) allele, the sequence of dtxR from the 1030(-) strain is only 91.4% identical; however, the deduced amino acid sequence of DtxR from 1030(-) differs by only 6 of 678 amino acids. Moreover, DtxR from all three strains is shown to regulate expression of beta-galactosidase from a tox promoter-operator (toxPO)-lacZ transcriptional fusion. In contrast, the dtxR allele from the iron-insensitive tox constitutive mutant C7hm723 was found to have a single G----A transition, resulting in a substitution of Arg-47 to His and the loss of tox regulatory activity in recombinant Escherichia coli.

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  1. Aisen P., Listowsky I. Iron transport and storage proteins. Annu Rev Biochem. 1980;49:357–393. doi: 10.1146/annurev.bi.49.070180.002041. [DOI] [PubMed] [Google Scholar]
  2. BARDSDALE W. L., PAPPENHEIMER A. M., Jr Phage-host relationships in nontoxigenic and toxigenic diphtheria bacilli. J Bacteriol. 1954 Feb;67(2):220–232. doi: 10.1128/jb.67.2.220-232.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  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. Fourel G., Phalipon A., Kaczorek M. Evidence for direct regulation of diphtheria toxin gene transcription by an Fe2+-dependent DNA-binding repressor, DtoxR, in Corynebacterium diphtheriae. Infect Immun. 1989 Oct;57(10):3221–3225. doi: 10.1128/iai.57.10.3221-3225.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. GUNDERSEN W. B. Investigation on the serological relationships of Corynebacterium diphtheriae, type mitis, and Corynebacterium belfanti. Acta Pathol Microbiol Scand. 1959;47:65–74. doi: 10.1111/j.1699-0463.1959.tb03422.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Kanei C., Uchida T., Yoneda M. Isolation from corynebacterium diphtheriae C7(beta) of bacterial mutants that produce toxin in medium with excess iron. Infect Immun. 1977 Oct;18(1):203–209. doi: 10.1128/iai.18.1.203-209.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kraft R., Tardiff J., Krauter K. S., Leinwand L. A. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques. 1988 Jun;6(6):544-6, 549. [PubMed] [Google Scholar]
  10. Maximescu P. New host-strains for the lysogenic Corynebacterium diphtheriae Park Williams No. 8 strain. J Gen Microbiol. 1968 Aug;53(1):125–133. doi: 10.1099/00221287-53-1-125. [DOI] [PubMed] [Google Scholar]
  11. Murphy J. R., Pappenheimer A. M., Jr, de Borms S. T. Synthesis of diphtheria tox-gene products in Escherichia coli extracts. Proc Natl Acad Sci U S A. 1974 Jan;71(1):11–15. doi: 10.1073/pnas.71.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Murphy J. R., Skiver J., McBride G. Isolation and partial characterization of a corynebacteriophage beta, tox operator constitutive-like mutant lysogen of Corynebacterium diphtheriae. J Virol. 1976 Apr;18(1):235–244. doi: 10.1128/jvi.18.1.235-244.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Pappenheimer A. M., Jr, Murphy J. R. Studies on the molecular epidemiology of diphtheria. Lancet. 1983 Oct 22;2(8356):923–926. doi: 10.1016/s0140-6736(83)90449-x. [DOI] [PubMed] [Google Scholar]
  15. Putnam S. L., Koch A. L. Complications in the simplest cellular enzyme assay: lysis of Escherichia coli for the assay of beta-galactosidase. Anal Biochem. 1975 Feb;63(2):350–360. doi: 10.1016/0003-2697(75)90357-7. [DOI] [PubMed] [Google Scholar]
  16. Rappuoli R., Michel J. L., Murphy J. R. Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox- into two attachment sites on the Corynebacterium diphtheriae chromosome. J Bacteriol. 1983 Mar;153(3):1202–1210. doi: 10.1128/jb.153.3.1202-1210.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ratti G., Rappuoli R., Giannini G. The complete nucleotide sequence of the gene coding for diphtheria toxin in the corynephage omega (tox+) genome. Nucleic Acids Res. 1983 Oct 11;11(19):6589–6595. doi: 10.1093/nar/11.19.6589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Schmitt M. P., Holmes R. K. Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae. Infect Immun. 1991 Nov;59(11):3903–3908. doi: 10.1128/iai.59.11.3903-3908.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Welkos S. L., Holmes R. K. Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production. J Virol. 1981 Mar;37(3):936–945. doi: 10.1128/jvi.37.3.936-945.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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