Complete Title of Thesis:
"Characterization of the Diphtheria-Toxin-Repressor (DtxR) Regulon in Corynebacterium diphtheriae
Prepared under the direction of: Randall K. Holmes, M.D., Ph.D.
In Corynebacterium diphtheriae, the diphtheria toxin repressor (DtxR) functions as an iron-dependent global regulator. We constructed a novel reporter transposon designated TnKnXSp, based on EZ-Tn5™<Kan-2> Tn, for studying gene expression and regulation in C. diphtheriae or other bacteria. This reporter transposon possesses a constitutively expressed spectinomycin resistance gene and a promoterless-kanamycin reporter gene positioned so that its expression depends on initiates from a bacterial promoter and proceeds into the integrated transposon from the appropriate end. We constructed transposon-insertion libraries in C. diphtheriae using this novel reporter transposon and used them to investigate the DtxR regulon. Among approximately 1,900 TnKnXSp insertion mutants screened for iron-regulated Kn-resistance phenotypes, we identified 38 mutants with transposon insertions in putative iron-repressed genes. Further analysis of these Kn-resistant mutants for specificity of their responses to iron versus other divalent metals identified three different phenotypic categories: 1) iron-specific, 2) regulated either by iron or by divalent metals other than iron, and 3) not regulated by iron or other metal ions tested. The sites of TnKnXSp insertions in these 38 mutants were determined, and DNA sequences contiguous to the insertion sites were examined for putative DtxR binding sites. Among these 38 mutants, we identified six previously reported and five novel iron- and DtxR-regulated genes. Seven mutants had putative DtxR-binding sites less than 300 base pairs upstream from the gene containing the TnKnXSp insertion, and five showed a putative DtxR binding site upstream from the first gene of a putative operon within which the transposon insertion was located. Four of the novel ironregulated mutants exhibited a siderophore-deficient phenotype, and further studies showed that DtxR was directly involved in regulating two of them. The fifth novel iron-regulated mutant has an insertion in a putative ClpB protease and exhibits a unique phenotype showing that DtxR is required for its expression under low-iron conditions. Therefore, this study has identified four novel genes involved in siderophore production, has added three new genes to the DtxR regulon and has provided the first evidence that DtxR regulates production of ClpB protease in C. diphtheriae.