BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 DtxR regulon of C. glutamicum since they lack DtxR bind- ing sites in their upstream regions. Investigating the phys- iological role of these genes in the cellular response to the subsequent addition of iron might eventually allow a bet- ter understanding of the molecular mechanisms used by C. glutamicum to counter high-iron conditions. Furthermore, the combination of bioinformatics predic- tions and DNA band shift assays resulted in the identifica- tion of DtxR binding sites within the upstream region of 31 genes and gene clusters, suggesting that at least 64 genes are under direct transcriptional control by DtxR in C. glutamicum. For a closer examination of the identified DtxR binding sites, transcriptional start sites of four differ- entially expressed genes were determined and the corre- sponding promoters were mapped. Three DtxR binding sites were found to overlap the deduced promoter regions, suggesting that DtxR acts as transcriptional repressor by blocking RNA polymerase binding and thus preventing expression of the respective genes [25]. Similar results were reported upon promoter mapping and localization of DtxR binding sites in C. diphtheriae [40] and of IdeR binding sites in M. smegmatis and M. tuberculosis [41,42]. On the other hand, the DtxR binding sites of cg2782, cg3327 and cg0445 were located upstream of the deduced -35 promoter region. Since the respective genes showed a decreased expression in C. glutamicum IB2103 during DNA microarray hybridization, this location is more con- sistent with an activating function of the corresponding transcriptional regulator [25]. In general, transcriptional regulators activate the expression of genes in such a way that they facilitate interaction of the RNA polymerase with the respective promoters. However, the large distance of the DtxR binding site to the -35 promoter region of the cg0445 gene cluster that encodes the iron-containing suc- cinate dehydrogenase complex of C. glutamicum suggests an alternative activation mechanism. In this context it is noteworthy that the cg0445 gene cluster is also under direct transcriptional control by the RipA repressor [23], a member of the AraC-family of DNA-binding transcrip- tional regulators [7]. Some members of the AraC protein family fulfill their regulatory function by using two DNA binding sites and a DNA looping mechanism to control gene expression [43]. The DtxR binding site in front of cg0445 is located directly adjacent to the RipA binding site A, and DtxR binding might therefore interfere with RipA binding to exert its regulatory function. Since expression of the ripA gene is also controlled by DtxR, the corre- sponding network topology of the DtxR regulon repre- sents a coherent feed-forward loop of type 4, in which the direct connection to the target gene is activating and the indirect connections are both repressing [44]. In such a way environmental input signals can be integrated into the transcriptional regulatory network at different levels of regulatory hierarchy to control expression of the succinate dehydrogenase complex. In addition to the cg0445 gene cluster, the dps (cg3327) and ftn (cg2782) genes showed a decreased expression in the dtxR mutant C. glutamicum IB2103. Dps-like proteins may function either as anti-redox agents or iron storage proteins, whereas ferritins act primarily in iron storage [13]. These data indicated that the DtxR protein can exert a regulatory role as positive modulator of expression of genes that are involved in iron storage and DNA protec- tion. This mechanism of transcriptional regulation is rea- sonable since the iron storage and protection genes are not expressed under low-iron conditions, whereas both, the iron storage capacity of the cell and the protection of DNA, increase under high-iron conditions by activating the respective genes. In M. tuberculosis, expression of the iron storage genes bfrA and bfrB, encoding a bacterioferri- tin and a ferritin-like protein, is induced also by iron and IdeR [36,42]. Binding of IdeR to the regulatory region of bfrA indicated that the gene is transcribed from a pro- moter that is activated by iron and binding of IdeR, since induction of bfrA gene expression was detected in the wild-type but not in an ideR mutant strain. The transcrip- tional control of iron storage in E. coli was also shown to be positively regulated by iron but no direct interaction of Fur with the regulatory region of the bfrA and ftn genes has been observed [45,46]. The comparative content analysis of DtxR binding sites in four corynebacterial genome sequences provided further insights into the topology of the DtxR regulon of C. glutamicum. The deduced consensus sequences of DtxR binding sites are not only very similar among the coryne- bacterial species but also resemble IdeR and DmdR1 bind- ing sites that were detected in M. tuberculosis and S. coelicolor, respectively [42,47]. Consequently, DtxR-like regulatory proteins utilize conserved recognition signals in different actinobacteria, as is the situation with DNA binding sites of the Fur repressor in gamma-proteobacte- ria [38]. On the other hand, the comparative content anal- ysis revealed that most of the DtxR-regulated genes are species-specific in the four sequenced corynebacteria, indicating that a wide variety of genetic information is used in the individual species to provide an effective iron homeostasis. Varying genetic information can be gained for instance by horizontal gene transfer, as suggested for the C. jeikeium genome that contains large sets of DtxR- regulated iron acquisition genes on mobile genetic ele- ments [10]. As iron metabolism is closely connected to iron toxicity, any additional genetic information regard- ing iron metabolism has to be integrated into the pre- existing DtxR regulon to avoid detrimental consequences for the bacterial cell. Only in case of similar DNA binding sites being present in the regulatory region of the respec- Page 14 of 19 (page number not for citation purposes)