BMC Genomics 2006, 7:21 http://www.biomedcentral.com/1471-2164/7/21 Table 2: Predicted DtxR binding sites in corynebacterial genome sequences (Continued) JK1780 JK1783 JK1805 JK1809 JK1812 JK1815 JK1819 JK18872 JK1934 JK1979 JK1983 ATAGGTTAGCCTACCCTTT TTAGGTTATGCTAAGTTAA TTAGGCAAGGGTAAGCTAA ACAGGTTAGGCTAACCAGA TTAGGTAAGGCTACCATCA CAAGTGAAGGCTTACCTTA CTTGATTAGCCTAACCTAA TTAAGCAAGGCTTGACTAA TAAGGTTCGGCTAACTGTA GCAGGTGACGCTAACCTGT GTAGCCTAGCCTTACCTAA GAAGGTGTGGCTAACCTAA - JK1782-1781 - JK1808-1806 - JK1816–1818 JK1820–1821 JK1886-1884 - - JK1984–1985 L-ornithine N5-oxygenase ABC transporter iron utilization protein ABC transport system solute binding protein iron ABC transport system siderophore synthesis system iron ABC transport system HTH_3-family regulatory protein ornithine cyclodeaminase iron ABC transport system - - - - - - - cg0926, CE0881 - - - 1 C. diphtheriae genes described previously as part of the DtxR regulon [14, 15] 2 Two DtxR binding sites were identified in front of the coding region. 3 A single DtxR binding site is involved most likely in regulation of adjacent coding regions located on the opposite DNA strand [14, 15]. constructed within the coding region of the dtxR gene, resulting in the mutant strain C. glutamicum IB2103 that was unable to grow in CGXII minimal medium under high-iron conditions. However, when the dtxR mutant C. glutamicum IB2103 was cultivated in low-iron CGXII medium, it revealed the same growth behavior as the wild-type control. These experiments showed that the dtxR gene is dispensable in C. glutamicum under specific culture conditions, as it was previously demonstrated for the orthologous regulatory genes dtxR, dmdR1 and ideR of C. diphtheriae, Streptomyces coelicolor and Mycobacterium smegmatis, respectively [33-35]. On the other hand, the ideR gene of Mycobacterium tuberculosis can be inactivated only in the presence of a second functional copy of the gene or when a second-site suppressor mutation alleviates the lethal effects of ideR inactivation, making ideR an essential gene in this bacterial species [36]. As observed with the defined dtxR mutant strain C. glutamicum IB2103, growth of the dtxR transposon mutant C7(β)18.5 of C. diphtheriae was dependent on the amount of iron that was added to the culture medium [33]. The C7(β)18.5 mutant of C. diphtheriae exhibited an extended lag phase and slower exponential growth in low-iron medium when compared with the parental C7(β) wild- type strain. Moreover, the C7(β)18.5 mutant grew only poorly in high-iron medium when compared with a con- trol medium containing no additional iron sources, and it was more-easily killed by exposure to high-iron condi- tions and H2O2 than was the parental strain [33]. Since the DtxR protein apparently regulates the expression of iron uptake systems, it might also be involved in protect- ing the cell from damage that is caused by high intracellu- lar iron concentrations. Transcriptional deregulation of iron homeostasis favors the Fenton reaction, leading to the production of hydroxyl radicals that can damage all biological macromolecules [37]. Therefore, transcrip- tional regulation of iron metabolism by the DtxR protein in C. glutamicum is not only necessary for economic rea- sons but also to avoid inevitable iron toxicity. In E. coli, iron homeostasis and oxidative stress response are closely interconnected and strictly controlled by the transcrip- tional regulator Fur [38]. Inactivation of the fur gene enhanced the sensitivity of the cell to redox stress, a phys- iological effect that can be reversed by iron chelation, by inhibiting ferric iron transport or by enhancing the iron storage capacity of the cell [37]. The dtxR mutant C. glutamicum IB2103 was further on used to compare the genome-wide expression pattern with that of the wild-type strain by DNA microarray hybridizations. In principle, inactivation of a gene encod- ing a repressor results in constitutive expression of those genes that are directly regulated by the respective protein. Therefore, comparative DNA microarray hybridizations are typically carried out in such a way that the relevant genes of the wild-type control are repressed and those of the mutant strain are de-repressed [39]. Since the dtxR mutant C. glutamicum IB2103 grew only in low-iron medium containing a trace amout of iron, the genes of the DtxR regulon are apparently de-repressed in both the mutant strain and the wild-type control. To detect differ- ential gene expression in C. glutamicum IB2103, we applied an alternative experimental setup by the subse- quent addition of an appropriate amount of iron to low- iron growth medium. Indeed, the DtxR repressor switched off the expression of its target genes in the wild-type strain, whereas the target genes were further on expressed in the dtxR mutant. This physiological effect was detecta- ble on a global scale by DNA microarray experiments and enabled the screening for differentially expressed genes that were later on assigned to the DtxR regulon of C. glutamicum. In addition to genes belonging to the DtxR regulon, the DNA microarray hybridization identified numerous coding regions whose expression was posi- tively or negatively influenced in response to the iron stimulus. These genes are considered to be subject to indi- rect regulation by iron and are apparently not part of the Page 13 of 19 (page number not for citation purposes)