Abstract
BackgroundThe knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum.ResultsA deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network.ConclusionThis work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the expression of genes involved in iron metabolism and exerts a dual regulatory function as repressor of genes participating in iron uptake and utilization and as activator of genes responsible for iron storage and DNA protection. The data suggest that the DtxR protein acts as global regulator by controlling the expression of other regulatory proteins that might take care of an iron-dependent regulation of a broader transcriptional network of C. glutamicum genes.
Highlights
The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks
To explore the regulatory network controlled by the DtxR protein of C. glutamicum, a defined deletion was established in the coding region of dtxR by applying a gene replacement strategy [19] that resulted in the mutant strain C. glutamicum IB2103
Growth assays with C. glutamicum IB2103 were performed in liquid CGXII minimal medium and growth of the cultures was monitored by nephelometry (Figure 1)
Summary
The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. One of the major challenges in post-genomic research is to decipher and reconstruct the complete connectivity of transcriptional regulatory networks encoded by a bacterial genome sequence [1,2]. The transcriptional network of the Gramnegative bacterium Escherichia coli is currently the bestunderstood regulatory system of a single cell. It includes 314 DNA-binding transcription factors and is characterized by a hierarchical and modular architecture that is composed of eight modules with distinct physiological functions. Even for this well-studied model organism, only a small fraction of the transcriptional regulatory interactions are currently known [3,5]
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