BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 glutamicum genome sequence as visualized by the GenDB database system [6]. This genome annotation contains bioinformatics predictions of Rho-independent transcrip- tional terminators that were generated by the sofware tool TransTerm [26]. An inspection of the genome sequence for the presence of transcriptional terminators in the downstream regions of genes that are regulated by DtxR finally resulted in the identification of twelve gene clusters or operons (Figure 5). Taking these bioinformatics predic- tions into account along with the functional annotation of the differentially expressed genes, at least 64 genes appeared to be directly regulated by the DtxR protein. The respective genes and gene clusters of the C. glutamicum genome as well as DNA microarray hybridization data regarding the differential expression in the dtxR mutant C. glutamicum IB2103 are shown in Figure 5. The genes belonging to the DtxR regulon of C. glutamicum can be divided into seven functional categories (Figure 5) according to the predicted physiological roles of the deduced proteins [6]. The functional classification yielded a prominent group of 26 genes that encode iron transport systems or siderophore interacting proteins. Accordingly, this functional category includes almost half of the genes that are under direct transcriptional control by DtxR. Additionally, the genes cg2445 (hmuO), cg2782 (ftn) and cg3327 (dps) were identified as part of the DtxR regulon of C. glutamicum. The respective proteins specify the heme oxygenase HmuO involved in the release of iron from the heme moiety [27], a putative ferritin most likely involved in iron storage [13] and a Dps homolog that can act either in iron storage or as DNA-protecting anti-redox agent [13]. Expression of the ftn and dps genes was decreased in the dtxR mutant C. glutamicum IB2103 as was the expres- sion of the sdh gene cluster that encodes the components of the iron-containing succinate dehydrogenase complex of C. glutamicum [6]. The genes belonging to other catego- ries of the DtxR regulon encode proteins without a precise functional prediction, secreted proteins of unknown func- tion or even hypothetical proteins (Figure 5), with the exception of the cgl gene cluster that represents the main DNA restriction and modification system of C. glutamicum ATCC 13032 [28] and the cysI-fpr2 gene region involved in assimilatory sulphate reduction [29]. Among the set of regulatory genes that are under direct control by DtxR, three DNA-binding transcriptional regu- lators were identified. This set includes the cg1120 (ripA) gene that was recently shown to be involved in iron metabolism of C. glutamicum [23] as well as the cg0527 and cg3082 genes encoding members of the ArsR family of regulatory proteins that typically exert metal-dependent transcriptional regulations in bacterial cells [7]. These data clearly indicate that the DtxR protein not only regu- lates the expression of genes playing a direct role in iron metabolism of C. glutamicum but also that of genes encod- ing transcriptional regulators, which can direct the expres- sion of further target genes at another level of regulatory hierarchy. Genome-wide identification of putative DtxR binding sites in corynebacterial genome sequences and comparative content analysis of the deduced DtxR regulons Since genes encoding a DtxR homolog are highly con- served in corynebacteria [30], the deduced genetic and functional composition of the DtxR regulon of C. glutami- cum was compared with DtxR regulons that were pre- dicted by bioinformatics analysis of the genome sequences of C. efficiens [8], C. diphtheriae [9] and C. jeikeium [10]. A multiple nucleotide sequence alignment of the 31 verified 19-bp motifs of C. glutamicum provided the seed information to create a HMM profile that was used to search for the presence of DtxR binding sites in the corynebacterial genome sequences. The resulting hits were applied to generate species-specific HMMs, and the genome sequences were searched again for the presence of potential DtxR binding sites with the respective profiles. This iterative approach resulted in the prediction of 15 DtxR binding sites in the genome sequence of C. efficiens, whereas 27 DtxR binding sites were detected in the genome sequence of C. diphtheriae and 21 in that of C. jeikeium (Table 2). With respect to the genome annota- tions and the bioinformatics prediction of gene clusters at least 27 genes might be under direct transcriptional con- trol by DtxR in C. efficiens, 59 in C. diphtheriae and 51 in C. jeikeium (Table 2). Moreover, the identified 19-bp motifs were used to gener- ate species-specific consensus sequences of the DtxR bind- ing sites by means of the WebLogo tool [31]. The resulting sequence logos are shown in Figure 6A. The overall height of each stack of letters indicates the sequence conservation at each position of the 19-bp motif, wheras the heigth of each symbol within the stack reflects the relative fre- quency of the corresponding nucleotide at that position. A comparison of the sequence logos clearly indicated that the four 19-bp motifs resemble the overall palindromic consensus sequence 5'-TTAGGTTAG(G/C)CTAACCTAA- 3' of DtxR binding sites, with apparent species-specific variations (Figure 6A). Accordingly, the sequenced corynebacterial genomes not only encode a conserved DtxR homolog but also contain sets of similar 19-bp motifs that represent potential DtxR binding sites. These data along with the verified DtxR binding sites of C. glutamicum and the annotated genome sequences of C. efficiens, C. diphtheriae and C. jeikeium enabled us to ana- lyze and compare the genetic composition of the DtxR regulons in these corynebacterial species. Page 8 of 19 (page number not for citation purposes)