ABSTRACTTranscription of the bacteriophage-borne diphtheria toxin genetoxis negatively regulated, in response to intracellular Fe2+concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis ofCorynebacterium diphtheriaehas primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments withC. diphtheriaegenes cloned inEscherichia colior analyzed in vitro. We modified a Tn5-based mutagenesis technique for use withC. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream ofdtxRand the other within an essential region of thedtxRcoding sequence, indicating for the first time thatdtxRis a dispensable gene inC. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-typeC. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion withindtxRdoes not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion withindtxRabolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.