BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 Background One of the major challenges in post-genomic research is to decipher and reconstruct the complete connectivity of transcriptional regulatory networks encoded by a bacterial genome sequence [1,2]. The key components in regula- tion of bacterial gene expression are DNA-binding tran- scription factors that are able to sense changing environmental conditions and to modulate the expres- sion of relevant target genes. An important prerequisite for understanding the regulation of gene expression in a bacterial cell in its entirety is the identification of the rep- ertoire of regulatory proteins encoded by a genome sequence along with the complete set of genes that are under transcriptional control by each of the identified reg- ulators [3,4]. The transcriptional network of the Gram- negative bacterium Escherichia coli is currently the best- understood regulatory system of a single cell. It includes 314 DNA-binding transcription factors and is character- ized by a hierarchical and modular architecture that is composed of eight modules with distinct physiological functions. Even for this well-studied model organism, only a small fraction of the transcriptional regulatory interactions are currently known [3,5]. The long-term objective of our post-genomic work is to characterize the complete repertoire of regulatory proteins of the Gram-positive bacterium Corynebacterium glutami- cum along with the complete set of their target genes and corresponding DNA binding sites within the known genome sequence [6,7]. Since we also want to understand the evolution of the transcriptional regulatory network of C. glutamicum, we took advantage of the published genome sequences of Corynebacterium efficiens [8], Coryne- bacterium diphtheriae [9] and Corynebacterium jeikeium [10] to perform comparative genomic analyses. C. glutamicum and C. efficiens are widely used in biotechnological fer- mentation processes, whereas C. diphtheriae and C. jeikeium represent the most hazardous human pathogens among the corynebacteria. Therefore, the four species rep- resent attractive targets to elucidate and compare not only the complete collection of DNA-binding transcriptional regulators but also the connectivity of regulatory interac- tions encoded by each genome sequence. In a recent study, we have determined the complete sets of DNA-binding transcriptional regulators of the four corynebacteria and have performed a comparative con- tent analysis of these genomes [7]. A collection of 127 DNA-binding transcriptional regulators was identified in the genome sequence of C. glutamicum, whereas 103 reg- ulators were identified in C. efficiens, 63 in C. diphtheriae and 55 in C. jeikeium. The common set of transcriptional regulators encoded by the four corynebacterial genomes consists of only 28 proteins. Considering functional assignments deduced from computational predictions the common DNA-binding transcriptional regulators were grouped into five modules with distinct physiological functions [7]. The functional module "macroelement and metal homeostasis" includes, for instance, the transcrip- tional regulator McbR, directing the global regulation of almost all aspects of sulphur metabolism, a FurB homolog, most likely involved in regulation of zinc metabolism [7], and a transcriptional regulator that is homologous to the diphtheria toxin repressor DtxR of C. diphtheriae [11]. The C. glutamicum DtxR protein shares 71% identical amino acid residues with the orthologous counterpart of C. diphtheriae. The diphtheria toxin repressor DtxR has been shown to be a global transcriptional regulator of iron metabolism in C. diphtheriae [12]. Iron is essential for a variety of cellular processes in virtually all organisms, since it plays impor- tant roles as enzyme cofactor or as integral part of cyto- chromes in oxidation-reduction and energy-generating systems [13]. On the other hand, iron poses problems in biological systems since the oxidized form is poorly solu- ble and the reduced form is highly toxic. Therefore, bacte- ria have evolved various mechanisms to counter these problems and to achieve an effective iron homeostasis. In addition, expression of the iron homeostatic machinery is subject to iron-dependent transcriptional regulation, for instance of genes encoding high-affinity iron uptake sys- tems, intracellular iron storage proteins, redox-stress resistance systems, and iron-containing proteins to con- trol overall iron consumption of the cell [13]. In C. diph- theriae, almost 20 binding sites of DtxR have been identified either by targeted genetic experiments or by a global repressor titration assay [14,15]. Major physiologi- cal functions that are controlled by DtxR in response to the iron level include the expression of the diphtheria toxin [12], the synthesis and export of siderophores [14], the siderophore-dependent uptake of iron [16], and the synthesis of specific systems for the utilization of iron from heme, hemin and hemoglobin [17,18]. In the present study, we have characterized the regulon of the transcriptional regulator DtxR of C. glutamicum by a genome-wide approach using DNA microarray technol- ogy. This global strategy along with bioinformatics predic- tions permitted the identification of DtxR binding sites in the C. glutamicum genome sequence along with a set of target genes that are under direct transcriptional control by DtxR. Binding of purified His-tagged DtxR protein to the upstream region of the respective genes was verified in vitro by DNA band shift assays. The resulting data were used for a comparative analysis of the deduced DtxR reg- ulons in the four sequenced corynebacterial species. Page 2 of 19 (page number not for citation purposes)