BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 technical replicates present on the two microarrays along with an error probability of less than 5% for the Student's t-test. Normalization of the hybridization data by the LOWESS function and t-test statistics were accomplished by the EMMA software package. The resulting ratio/inten- sity (m/a) plot of the normalized data is presented in Fig- ure 2. By applying a ratio cut-off of ± 1, corresponding to relative expression changes equal or greater than twofold, a total of 257 genes revealed a differential expression in C. glutamicum IB2103 (see Additional file 1). This number includes 131 genes with significantly increased expression in C. glutamicum IB2103 when compared to the wild-type strain (m-value equal or greater than +1.0) and 126 genes with decreased expression in the dtxR mutant (m-value equal or smaller than -1.0). Several genes showing dere- pression of transcription in C. glutamicum IB2103 when compared to the wild-type strain encode iron-containing proteins and proteins that are apparently involved in iron transport and metabolism (Figure 2). Consequently, the upstream regions of the respective genes were ideally suited to search for the presence of a common DtxR bind- ing motif. Computational identification of potential DtxR binding sites in the genome sequence of C. glutamicum ATCC 13032 To identify DtxR binding sites in the genome sequence of C. glutamicum, the upstream regions of 13 differentially expressed genes encoding proteins involved in iron metabolism were selected (Table 1) and aligned by using the CLUSTAL X program [21]. The alignment identified a common 19-bp DNA motif with the palindromic consen- sus sequence 5'-TTAGGTTAG(G/C)CTAACCTAA-3' that is identical to DtxR binding sites identified earlier in C. diph- theriae [14]. A CLUSTAL X alignment of the 19-bp motifs was used to create a Hidden Markov model (HMM), and the C. glutamicum genome sequence was searched for the presence of additional motifs with this HMM profile. The results generated by HMM searches were then compared with the genomic positions of genes that showed differen- tial expression in the dtxR mutant C. glutamicum IB2103 during DNA microarray hybridization. This bioinformat- ics approach identified 24 motifs in front of genes that were identified as differentially expressed by DNA micro- array hybridization, including the 13 motifs initially used as seed information to create the HMM profile (Table 1). It is noteworthy that three 19-bp motifs were predicted in front of genes (cg2782/ftn, cg3327/dps, cg0445/sdhCD) that showed a decreased expression in the dtxR mutant C. glutamicum IB2103 (Table 1). The bioinformatics search identified additional 16 motifs upstream of genes that were not detected during the DNA microarray hybridiza- tion experiment. These motifs either represent false-posi- tive predictions or might be related to genes that were not detected with significant values during DNA microarray hybridization (see below). Accordingly, a total number of 40 potential DtxR binding sites was identified in the C. glutamicum genome sequence by bioinformatics analysis and further on investigated experimentally. Especially genes that showed an enhanced or decreased expression in the dtxR mutant C. glutamicum IB2103 and that are moreover characterized by the presence of a predicted binding site within the corresponding upstream region can be considered as candidates for direct transcriptional regulation by DtxR. Verification of differential expression of genes with predicted DtxR binding sites by real-time RT-PCR experiments Differential expression of the 40 genes identified by bio- informatics screening of the C. glutamicum genome sequence for the presence of DtxR binding sites was exam- ined further by real-time RT-PCR assays using the same experimental setup as applied for DNA microarray hybrid- izations. The resulting data are summarized in Table 1. The real-time RT-PCR assays demonstrated that the expression of 31 genes was enhanced up to 2000-fold in the dtxR mutant C. glutamicum IB2103 when compared with the wild-type strain. Accordingly, the results can be divided into three categories: (i) Differential expression in C. glutamicum IB2103 of the 24 genes initially identified by the DNA microarray experiment was confirmed by the RT-PCR approach. (ii) Among the genes additionally pre- dicted by HMM searches seven showed indeed a differen- tial expression in the dtxR mutant strain C. glutamicum IB2103. Expression of four genes was enhanced only up to 5-fold, and a further examination of the DNA microarray data indicated that at least two of the respective ratios were close to the experimental cut-off (data not shown). (iii) In the case of nine potential motifs, the prediction by the HMM profile was most likely false-positive, since the corresponding genes showed no significant changes of the expression level in C. glutamicum IB2103, neither by DNA microarray hybridization nor by real-time RT-PCR meas- urements. Consequently, the combination of DNA micro- array hybridization, bioinformatics screening approaches and real-time RT-PCR assays led to a set of genes that are differentially expressed in the dtxR mutant C. glutamicum IB2103 and that are characterized by a putative DtxR binding site within the upstream region. Binding of purified DtxR protein to the identified sequence motifs as analyzed by DNA band shift assays To demonstrate binding of the DtxR regulator to the pre- dicted 19-bp motifs by DNA band shift assays, a modified DtxR protein was constructed and subsequently purified. For this purpose, the coding region of the dtxR gene was fused with a 3' extension encoding six histidine residues Page 6 of 19 (page number not for citation purposes)