Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength

doi:10.1128/JB.182.2.432-438.2000

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Journal of Bacteriology, January 2000, p. 432-438, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength

John H. Lee and Randall K. Holmes^*

Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received 10 May 1999/Accepted 27 October 1999

The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe²⁺ as a corepressor. Holo-DtxR inhibits transcription from the iron-regulatedpromoters (IRPs) designated IRP1 through IRP5 as well as fromthe promoters for the tox and hmuO genes. DtxR binds to 19-bpoperators with the consensus sequence 5'-TTAGGTTAGCCTAACCTAA-3',a perfect 9-bp palindrome interrupted by a single C · G base pair.Among the seven known DtxR-specific operators, IRP3 exhibits theweakest binding to DtxR. The message (sense) strand of the IRP3operator (5'-TTAGGTGAGACGCACCCAT-3' [nonconsensus nucleotidesunderlined]) overlaps by 2 nucleotides at its 5' end with theputative $-$ 10 sequence of the IRP3 promoter. The underlined C atposition +7 from the center of the IRP3 operator [C(+7)] is unique,because T is conserved at that position in other DtxR-specificoperators. The present study examined the effects of nucleotidesubstitutions at position +7 or $-$ 7 in the IRP3 operator. In gelmobility shift assays, only the change of C(+7) to the consensusnucleotide T caused a dramatic increase in the binding of DtxR,whereas other nucleotide substitutions for C(+7) or replacementsfor A( $-$ 7) had only small positive or negative effects on DtxRbinding. All substitutions for C(+7) or A( $-$ 7) except for A( $-$ 7)Cdramatically decreased IRP3 promoter strength. In contrast, theA( $-$ 7)C variant caused increased promoter strength at the costof nearly eliminating repressibility by DtxR. The message (sense)strand of the IRP1 operator (5'-TTAGGTTAGCCAAACCTTT-3') includesthe $-$ 35 region of the IRP3 promoter. A T(+7)C variant of the IRP1operator was also constructed, and it was shown to exhibit decreasedbinding to DtxR, decreased repressibility by DtxR, and increasedpromoter strength. The nucleotides at positions +7 and $-$ 7 in DtxR-specificoperators are therefore important determinants of DtxR bindingand repressibility of transcription by DtxR, and they also havesignificant effects on promoter activity for IRP3 andIRP1.

^* Corresponding author. Mailing address: Department of Microbiology, Campus Box B-175, University of Colorado Health SciencesCenter, 4200 East Ninth Ave., Denver, CO 80262. Phone: (303) 315-7903.Fax: (303) 315-6785. E-mail: Randall.Holmes{at}UCHSC.Edu.

Present address: Chonbuk National University, College of Veterinary Medicine, Chonju, 561-756, SouthKorea.

Journal of Bacteriology, January 2000, p. 432-438, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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