ABSTRACTPrevious studies withCorynebacterium diphtheriaeandMycobacteriumspecies revealed that the transcriptional regulator DtxR and its ortholog IdeR play a central role in the control of iron metabolism. In the present work, we used genome-based approaches to determine the DtxR regulon ofCorynebacterium glutamicum, a nonpathogenic relative ofC. diphtheriae. First, global gene expression of adtxRdeletion mutant was compared with that of the wild type using DNA microarrays. Second, we used a computer-based approach to identify 117 putative DtxR binding sites in theC. glutamicumgenome. In the third step, 74 of the corresponding genome regions were amplified by PCR, 51 of which were shifted by the DtxR protein. Finally, we analyzed which of the genes preceded by a functional DtxR binding site showed altered mRNA levels in the transcriptome comparison. Fifty-one genes organized in 27 putative operons displayed an increased mRNA level in theΔdtxRmutant and thus are presumably repressed by DtxR. The majority of these genes are obviously involved in iron acquisition, three encode transcriptional regulators, e.g., the recently identified repressor of iron proteins RipA, and the others encode proteins of diverse or unknown functions. Thirteen genes showed a decreased mRNA level in theΔdtxRmutant and thus might be activated by DtxR. This group included thesufoperon, whose products are involved in the formation and repair of iron-sulfur clusters, and several genes for transcriptional regulators. Our results clearly establish DtxR as the master regulator of iron-dependent gene expression inC. glutamicum.