BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 many). Standard CGXII minimal medium (10 mg/l FeSO4) and low-iron CGXII medium (0 mg/l FeSO4) were used for growth assays [33]. Growth of shaking flask cul- tures was monitored by measuring the optical density at 600 nm with an Eppendorf BioPhotometer. E. coli DH5αMCR was used for standard cloning procedures and grown at 37°C in Luria-Bertani medium [49] supple- mented with 2 g/l glucose. Selection for the presence of plasmids was performed with kanamycin (50 µg/ml for E. coli and 25 µg/ml for C. glutamicum). Isopropyl beta-D- thiogalactoside (IPTG) was used to induce dtxR expres- sion by the Ptrc promoter of pEC-XK99E [22]. DNA isolation, manipulation and transfer Vector DNA was prepared from E. coli cells by an alkaline lysis technique using the QIAprep Spin Miniprep Kit (Qia- gen, Hilden, Germany). Chromosomal DNA of C. glutamicum was prepared as described previously [51]. Modification of DNA, analysis by agarose gel electro- phoresis and ligation were performed by standard proce- dures [49]. Transformation of E. coli and C. glutamicum cells was performed by electroporation [52,53]. PCR techniques and dtxR mutant construction PCR experiments were carried out with a PTC-100 ther- mocycler (MJ Research, Watertown, MA), Pwo DNA polymerase (Roche Diagnostics, Mannheim, Germany) and chromosomal C. glutamicum DNA as template. PCR products were purified by using the PCR Purification Spin Kit (Qiagen). Oligonucleotides used for PCR amplifica- tion were purchased from Operon Biotechnologies (Cologne, Germany). The gene SOEing method [54] was applied to construct a pK18mobsacB derivative that is suit- able to perform an allelic exchange of the dtxR gene in the chromosome of C. glutamicum ATCC 13032 [19]. The primers used were cg2103del1 (GATCTAGAATTCCCAAG- GCGTGAGATGACAG), cg2103del2 (GAGCACGCAGAG- GAACAAT), cg2103del3 (ATTGTTCCTCTGCGTGCTCTCAAGCAGATGAGCCT- GAT), and cg2103del4 (GATCTAGGATCCTTCTACGCG- GACTGCATGT). The DNA fragment was digested with EcoRI and BamHI and cloned into the vector pK18mobsacB. The resulting plasmid pIB2103 carries a modified dtxR gene that is specified by a defined deletion of 338 nucleotides. Gene replacement in the chromosome of C. glutamicum ATCC 13032 resulted in the dtxR mutant strain C. glutamicum IB2103. For genetic complementa- tion of mutant strain C. glutamicum IB2103, the dtxR cod- ing region was amplified by PCR with the primer pair compl1 (GATCTAGAATTCAAAGGAGGACAACCAT- GAAGGATCTGGTCGATAC) and compl2 (GATCTAG- GATCCGTGTGTTAGCCCTCAACC). The PCR product was digested with EcoRI and BamH and cloned into the compatible sites of the IPTG-inducible expression vector pEC-XK99E [22]. The resulting plasmid pIB4000 was transferred into C. glutamicum ATCC 13032 and C. glutamicum IB2103 by electrotransformation. Total RNA preparation and DNA microarray hybridization For the preparation of total RNA, C. glutamicum cultures were grown in minimal medium CGXII. Approximately 1 × 109 cells from exponentially growing cultures were har- vested by centrifugation with 11,000 g for 15 s and subse- quently transferred into liquid nitrogen. RNA isolation, cDNA synthesis, labeling of probes, and DNA microarray hybridization were performed as described previously [20]. The experiments were carried out in duplicate using label swapping. Normalization and evaluation of data was accomplished by the EMMA microarray data analysis software using a m-value cut-off of ± 1, which corresponds to relative expression changes equal or greater than two- fold [20]. Since each DNA microarray contains four repli- cates per gene, a total number of eight spots per gene was available for calculating differential gene expression. Bioinformatics tools for DtxR binding site prediction The annotated version of the C. glutamicum ATCC 13032 genome sequence [6] was used to perform a genome-wide screening for putative DtxR binding sites. The search was accomplished by Hidden Markov model (HMM) analysis using the HMMER software package [55]. A set of pre- dicted DtxR binding sites was aligned by means of the CLUSTAL X program [21], and the resulting alignment was used to create a HMM by using the HMMBUILD mod- ule. The calculated HMM profile along with the HMMSEARCH module was applied to screen the C. glutamicum genome sequence for the presence of DtxR binding sites. The genomic positions of the resulting hits were correlated with coding sequences that revealed dif- ferential expression in the dtxR mutant when compared with the wild-type strain by DNA microarray hybridiza- tion. The experimentally verified DtxR binding sites of C. glutamicum were aligned with the CLUSTAL X program and used to generate a HMM profile for an iterative screening of the genome sequences of C. efficiens [8], C. diphtheriae [9] and C. jeikeium [10]. Real-time reverse transcription (RT)-PCR Purified total RNA of C. glutamicum cultures was used for real-time RT-PCR experiments with the LightCycler instru- ment (Roche Diagnostics) and the Quanti-Tect SYBR Green RT-PCR Kit (Qiagen). Oligonucleotides used to measure relative gene expression were purchased from Operon Biotechnologies. Verification of RT-PCR products was performed by melting curve analysis. Differences in gene expression were determined by comparing the cross- ing points of two samples measured in duplicate. Cross- ing points were calculated by the LightCycler software (Roche Diagnostics). Page 16 of 19 (page number not for citation purposes)