BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 and amplified by PCR. The resulting DNA fragment was cloned in E. coli into the expression vector pEC-XK99E [22] resulting in plasmid pIB4001 that is suitable for het- erologous overexpression of the modified DtxR protein upon IPTG induction. The His-tagged DtxR protein was purified to homogeneity from an induced E. coli culture by means of Ni-TED columns and positively identified by MALDI-TOF mass spectrometry and peptide mass finger- printing (data not shown). Moreover, plasmid pIB4001 was transferred into the dtxR mutant C. glutamicum IB2103 by electroporation to prove that the His-tagged DtxR protein is functional in vivo. This control experiment demonstrated that the growth deficiency of the dtxR mutant C. glutamicum IB2103 in CGXII minimal medium can be complemented by plasmid pIB4001 but not by the empty cloning vector pEC-XK99E (data not shown). Subsequently, DNA band shift assays were carried out with the purified His-tagged DtxR protein and distinct DNA fragments containing each of the 31 predicted 19-bp motifs. For this purpose, the motifs were extended on both sides with the respective gene-specific sequences to a total length of 40 nucleotides. These 40 mer oligonu- coletides were labeled with Cy3 and annealed to produce double-stranded DNA fragments that were applied in band shift assays. First of all, 0.05 pmol of the labeled 40 mer DNA fragment representing the predicted DtxR bind- ing site upstream of cg0771 was incubated with different amounts of His-tagged DtxR protein (42 pmol, 84 pmol, 126 pmol, 168 pmol, and 210 pmol). The assays were then separated by agarose gel electrophoresis and visual- ized by fluorescence imaging (Figure 3A). Apparently, the presence of purified DtxR protein caused a different elec- trophoretic mobility of the labeled DNA fragment even at the low protein concentration of 42 pmol. Furthermore, DNA band shift assays were performed with labeled 40 mer control fragments located within the coding region of cg0397 and cg0738 (dnaE2), respectively. Incubation of purified DtxR protein with these DNA fragments delivered no band shift (Figure 3B). Binding of the DtxR protein to the upstream region of cg0771 was also analyzed by DNA displacement experiments, in which increasing concentra- tions of the same non-labeled 40 mer fragment (0.015 to 1 pmol) were added to the band shift assay. Increasing concentrations of this sequence-specific competitor pre- vented the DNA band shift caused by the purified DtxR protein (Figure 3C). These data suggested a specific bind- ing of the DtxR regulator to the upstream region of cg0771 containing the common 19-bp motif. Subsequent DNA band shift assays were performed with 42 pmol of purified DtxR protein (Figure 3D). The pres- ence of purified DtxR protein caused a band shift of almost all tested 40 mer DNA fragments, with exception of a 40 mer region located in front of the cg1120 gene that encodes the ArsR-type regulatory protein RipA [23]. The reason for this observation is currently unknown since binding of the DtxR protein to the upstream region of cg1120 was described earlier [23]. Nevertheless, the DNA band shift assays confirmed in vitro the binding of the DtxR regulator to specific DNA fragments containing the common 19-bp motif that was identified in the upstream region of differentially expressed genes. Localization of DtxR binding sites in mapped promoter regions of differentially expressed genes To determine the position of verified DtxR binding sites in relation to the promoter region of the respective genes, transcriptional start sites were identified by using the 5' RACE method. Transcriptional starts were determined for the genes cg0771, cg0922 and cg0527 that showed an enhanced expression in the dtxR mutant C. glutamicum IB2103 and for the cg2782, cg3327 and cg0445 genes that revealed a decreased expression. The detected transcrip- tional start sites were identified in variable distances to the annotated translational starts of the corresponding pro- teins (Figure 4). The transcriptional start sites of cg0527, cg2782 and cg3327 were identical to the adenine residue of the annotated translational start codons. These results provided evidence for the presence of leaderless tran- scripts that are known to occur in C. glutamicum [24]. Moreover, the determination of transcriptional start points provided the basis for the identification of poten- tial -35 and -10 promoter regions, according to the known features of corynebacterial promoter sequences [24]. At least three nuclotides of the potential -35 and -10 hexam- ers were identical to the consensus sequences TTGCCA and TA(C/T)AAT, respectively. In the case of cg0771, cg0922 and cg0527 the verified DtxR binding sites were located within the deduced promoter region (Figure 4), which is consistent with the physiological role of the DtxR protein as transcriptional repressor of gene expression. On the other hand, the DtxR binding sites of cg2782, cg3327 and cg0445 were located upstream of the deduced -35 pro- moter regions (Figure 4). Since expression of these genes decreased in the dtxR mutant C. glutamicum IB2103, as measured by DNA microarray hybridization and real-time RT-PCR, the location of the DtxR binding sites would be more consistent with an activating function of the DtxR protein [25]. Bioinformatics characterization and functional dissection of the DtxR regulon of C. glutamicum The 31 genes, which were characterized by differential expression pattern in the dtxR mutant C. glutamicum IB2103 and by the presence of a DtxR binding site in front of the coding region, only represent a part of the DtxR reg- ulon since operon structures also have to be considered. Therefore, the genetic organization of the respective gene regions was analyzed by using the annotation of the C. Page 7 of 19 (page number not for citation purposes)