Genome-wide analysis of DtxR and HrrA regulons reveals novel targets and a high level of interconnectivity between iron and heme regulatory networks in Corynebacterium glutamicum

Abstract

Iron is an essential trace element required by nearly all organisms as a cofactor in enzymes, regulatory proteins, and cytochromes of the respiratory chain. Maintaining iron homeostasis is crucial, since elevated levels cause oxidative stress through the formation of reactive oxygen species. In Corynebacterium glutamicum, iron and heme homeostasis are tightly interconnected and controlled by the global regulators DtxR and HrrA. While DtxR senses intracellular Fe2+, the two-component system HrrSA is activated by heme, functioning as a global regulator of heme homeostasis. This study provides the first genome-wide analysis of DtxR and HrrA binding dynamics under varying iron and heme conditions using chromatin affinity purification sequencing (ChAP-seq). Our conditional ChAP- Seq approach revealed 25 novel DtxR targets and 210 previously unrecognized HrrA targets. Among these, metH, encoding homocysteine methyltransferase, and xerC, encoding a tyrosine recombinase, were bound by DtxR exclusively under heme conditions, underscoring condition-dependent variation in DtxR binding. Activation of metH by DtxR links iron metabolism to methionine synthesis, potentially relevant for the mitigation of oxidative stress. Beyond novel targets, this study highlights the interconnected nature of the DtxR and HrrA regulons, identifying 16 shared targets with in some cases overlapping operator sequences. Strikingly, we provide several examples for weak ChAP-Seq peaks, often disregarded in global approaches, that feature a significant impact of the regulator on differential gene expression. These findings emphasize the importance of genome-wide profiling under different conditions to uncover novel targets and shed light on the complexity and dynamic nature of bacterial regulatory networks. ImportanceThe trace element iron is essential for life, but elevated levels can rapidly cause cellular damage through oxidative stress. Bacteria, like Corynebacterium glutamicum, tightly regulate iron and heme homeostasis via the global regulators DtxR and HrrA. This study provides the first analysis of the genome-wide binding patterns of these two regulators demonstrating significant differences in binding dependent on the tested iron regimes. Overall, we identified 25 new DtxR targets and 210 previously unknown HrrA targets, including genes with crucial roles in central metabolism and DNA repair. Notably, DtxR was shown to link iron metabolism to methionine synthesis, which might be important to protect the cell from oxidative stress. Our findings highlight the interconnected nature of DtxR and HrrA networks and underscore the value of condition-specific analysis to deepen the understanding of how bacteria adapt to environmental changes.