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

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 87:5968-5972, 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 1-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.We have recently described the use of a diphtheria tox promoter-operator (toxPO)-lacZ transcriptional fusion in the molecular cloning of an iron-dependent tox regulatory element (dtxR) from Corynebactenum diphtheriae (4). DtxR is a 25,316-Da protein which was shown to be an iron-dependent negative control element in the expression of 3-galactosidase from the toxPO-lacZ fusion in recombinant Escherichia coli. This study was recently confirmed and extended by Schmitt and Holmes (25), who demonstrated that dtxR controlled the iron-dependent expression of tox and the siderophore genes in C. diphtheriae.Different strains of C. diphtheriae which are lysogenic for tox+ corynebacteriophage may under optimal conditions produce different yields of diphtheria toxin. The final yield of toxin produced may be influenced by the ability of a given strain to continue growth following exhaustion of iron from the medium (18) and the number of integrated tox+ corynebacteriophage genomes (22). Regardless of the final yield of toxin, the expression of the tox structural gene in C. diphtheriae is repressed by the addition of iron to the culture medium.In contrast, the expression of diphtheria toxin from the C7hm723(,tox+) strain of C. diphtheriae is insensitive to iron-mediated repression (11). The mutant C7hm723 strain was isolated after nitrosoguanidine mutagenesis and was postulated to carry a defect in the diphtheria tox regulatory gene. In addition, Cryz et al. (6) were able to demonstrate that, relative to the parental C7 strain, C7hm723 also had a decreased rate of iron uptake. In order to gain additional insight into the regulation of diphtheria tox expression in both wild-type and mutant strains of C. diphtheriae, the dtxR alleles of the PW8(-), 1030(-), and C7hm723(-) strains were cloned and sequenced and the tox regulatory activity of * Corresponding author. t Present address:

Metal stoichiometry and functional studies of the diphtheria toxin repressor

“…DtxR is a 226-amino acid polypeptide, which functions as a homodimer (8)(9)(10)(11). Recombinant DtxR was tested in vitro for interaction with DNA containing the tox operator region by gel electrophoretic mobility-shift assays (EMSAs) and DNase I and hydroxyl radical footprinting (12)(13)(14)(15).…”

Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron

“…In C. diphtheriae C7(,B)hm723, which carries a defective dtxR allele (2,16,32), diphtheria toxin and siderophore are produced constitutively at high levels, regardless of the iron concentration (5,31,37). However, when the cloned dtxR+ allele is introduced into C7(3)hm723, iron-dependent regulation of the phage-encoded tox gene and the chromosomally encoded determinants for siderophore production and utilization is restored (31 …”

Molecular Characterization of Diphtheria Toxin Repressor (dtxR) Genes Present in NontoxigenicCorynebacterium diphtheriaeStrains Isolated in the United Kingdom

“…DtxR variants 1, 4, and 5 were seen in the 72 isolates analyzed by Nakao et al (17). As reported previously, mutant strain C7hm723, which produces siderophore under high-and low-iron conditions, contains a single nucleotide substitution resulting in substitution of histidine for arginine at residue 47 of DtxR; this single amino acid substitution is sufficient to almost abolish the regulatory activity of DtxR from strain C7hm723 (2,30). The dtxR allele from strain PW8 is identical to that from strain C7(Ϫ), but dtxR from C. diphtheriae 1030 biotype Belfanti is only 91.7% identical to the allele from C7(Ϫ).…”

“…The dtxR allele from strain PW8 is identical to that from strain C7(Ϫ), but dtxR from C. diphtheriae 1030 biotype Belfanti is only 91.7% identical to the allele from C7(Ϫ). Strain 1030 has 56 base substitutions and encodes a DtxR variant that differs from C7(Ϫ) DtxR by 6 amino acid substitutions in the carboxyl-terminal region (I165V, V174A, S191T, S205R, L214Y, and L218T) (2). The amino-terminal half includes the DNA-binding motif and the metal-binding motif (37 (37).…”