BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 Construction and purification of a His-tagged DtxR protein A PCR product fusing the coding region of the C. glutami- cum dtxR gene with a nucleotide sequence encoding a C- terminal His tag was generated by using the primer pair dtxR-His1 (GATCTAGAATTCAAAGGAGGACAACCAT- GAAGGATCTGGTCGATA) and dtxR-His2 (GATCTAG- GATCCTTAATGGTGATGGTGATGGTGGCCCTCAACCTT TTCTAC). The PCR product was digested with EcoRI and BamHI and cloned into the IPTG-inducible expression vector pEC-XK99E [22]. The resulting plasmid pIB4001 was transferred to E. coli DH5αMCR and to C. glutamicum strains by electroporation. To isolate the His-tagged DtxR protein, E. coli DH5αMCR (pIB4001) was grown for 16 h in Luria-Bertani medium containing 50 µg/ml kanamycin and 0.1 mM IPTG. Approximately 4 × 1010 cells were har- vested by centrifugation, resuspended in LEW buffer (50 mM NaH2PO4, 300 mM NaCl, one tablet of Roche Com- plete Mini protease inhibitors; pH 8.0) and transferred into a RiboLyser tube (Hybaid, Heidelberg, Germany). Cell disruption by means of the RiboLyser instrument was carried out with a speed rate of 6.5 and two time intervals of 30 s. The His-tagged DtxR protein was purified from the protein crude extract with Protino Ni-TED 1000 packed columns (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. After loading of the protein crude extract, the resin column was washed twice with 2 ml LEW buffer. The His-tagged DtxR protein was eluted with 2.5 ml LEW buffer containing 10 mM imida- zole and finally stored at -20°C. The resulting eluate was concentrated by using Amicon ultra-4 5000 MWCO cen- trifugal filter units (Millipore, Schwalbach, Germany) and subsequently analyzed by SDS-PAGE [53]. The protein concentration was determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Munich, Germany). To verify the purification of the His-tagged DtxR protein, an aliqout of the eluate was enzymatically digested with modified trypsin (Promega, Mannheim, Germany) and the resulting peptide mass fingerprint was determined by MALDI-TOF mass spectrometry, applying an Ultraflex mass spectrometer (Bruker Daltonics, Bremen, Germany) and the MASCOT software. DNA band shift assays to characterize binding of the purified DtxR protein DNA band shift assays were performed with Cy3-labeled 40 mer oligonucleotides (Operon) that were annealed with corresponding complementary oligonucleotides to double-stranded DNA fragments by heating at 94°C for 5 min and annealing on ice for 15 min. During band shift assays, 42 pmol of purified His-tagged DtxR protein were mixed with 0.05 pmol DNA, 200 µM CoCl2, 15% (v/v) glycerol, and DtxR binding buffer (20 mM Na2HPO4, 50 mM NaCl, 5 mM MgCl2, 2 nM DTT, 10% (v/v) glycerol, 100 µg/ml BSA; pH 7.0 [56]), to get a total volume of 20 µl. The assay was incubated at room temperature for 15 min and then separated with a 2% agarose gel (containing 150 mM CoCl2) prepared in gel buffer (20 mM Na2HPO4; pH 7.0). A voltage of 80 V was supplied for 1.5 h. The aga- rose gel was scanned with a Typhoon 8600 Variable Mode Imager (Amersham Biosciences Europe, Freiburg, Ger- many). During displacement experiments, increasing con- centrations of non-labeled competitor DNA (0.05 to 1 pmol) were added to the reaction batch. Identification of transcriptional start sites by the RACE method For the identification of transcriptional start sites, total RNA was isolated from C. glutamicum wild-type cultures grown in CGXII medium. RACE primers (18 mer oligonu- cleotides) binding 200 to 300 nucleotides downstream of the annotated translational starts of investigated genes along with 1 µg of total RNA were used for cDNA synthe- sis. RACE primer sequences will be provided by the authors upon request. The resulting cDNA was modified and amplified by two additional PCRs using the 5'/3' RACE Kit second generation (Roche Diagnostics). PCR products were cloned into the pCR2.1-TOPO vector (Inv- itrogen, Karlsruhe, Germany) and transferred into compe- tent E. coli TOP10 cells. DNA sequencing of cloned RACE products was performed by IIT Biotech (Bielefeld, Ger- many). Authors' contributions IB carried out the experimental work and drafted the man- uscript. HW participated during real-time RT-PCR and RACE PCR experiments. ATH provided the DNA microar- ray. JK participated in data evaluation. AP conceived of the design of the figures and participated in supervision. AT conceived of the study and participated in co-ordination and supervision. All authors read and approved the final manuscript. Additional material Additional file 1 C. glutamicum genes transcriptionally up-regulated or down-regulated in the dtxR mutant IB2103 upon iron addition to the growth medium when compared to the wild-type ATCC 13032. Relevant molecular and expres- sion data of all genes detected as differentially expressed in C. glutami- cum IB2103 upon addition of iron to the growth medium Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-21-S1.pdf] Acknowledgements The authors thank Carola Eck for excellent technical assistance during MALDI-TOF mass spectrometry and Karina Brinkrolf for valuable discus- sions. Page 17 of 19 (page number not for citation purposes)