Novel insights into the transcriptional regulation of cell division in Corynebacterium glutamicum

Kim Julia Kraxner

In the first part of this doctoral thesis the transcriptional regulation of the odhI gene (cg1630) of Corynebacterium glutamicum was analyzed. OdhI in its unphoshorylated state functions as inhibitor of the 2-oxoglutarate dehydrogenase complex (ODHC) by binding to the OdhA subunit. Phosphorylation of OdhI by serine/threonine protein kinases abolishes this effect. Inhibition of ODHC activity by OdhI was shown to be crucial for overproduction and secretion of L-glutamate, which is used as a flavour enhancer. Since downstream of odhI two genes presumably encoding transcriptional regulators (cg1631 and cg1633) are located, it was speculated that these could be involved in transcriptional regulation of odhI. However, transcriptome analysis of deletion mutants lacking cg1631 or cg1633 and DNA affinity chromatography with the odhI promoter did not support this hypothesis. Furthermore, no other potential transcriptional regulators of odhI could be identified. Thus, there is currently no evidence for transcriptional regulation of odhI. The second part of this thesis addresses the regulation of cytokinesis in C. glutamicum. In contrast to e.g. Escherichia coli and Bacillus subtilis, knowledge about regulators of cytokinesis in Actinobacteria is very limited. In this study, the so far uncharacterized Cg1631 protein was discovered to be a transcriptional regulator of the ftsZ gene in C. glutamicum encoding the key player of bacterial cell division. Therefore, Cg1631 was named FtsR, standing for FtsZ regulator. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology, emphasizing an important function of FtsR in cell division or cell wall synthesis. The wild-type phenotype could be restored by plasmid-based complementation. Chromatin affinity purification with subsequent next generation sequencing (ChAP-Seq) identified a region in the ftsZ promoter as a major FtsR binding site, but revealed also additional potential target genes. With the ChAP-Seq results a putative DNA-binding motif could be identified for FtsR. Transcriptional activation of ftsZ expression by FtsR was underlined by DNA microarray experiments, electrophoretic mobility shift assays (EMSAs), and reporter gene studies. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ftsR mutant is not solely caused by reduced ftsZ expression but involves additional factors. In summary, FtsR was identified as the first transcriptional regulator of ftsZ in C. glutamicum. Furthermore, since FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, it can be assumed that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria. This makes FtsR a promising target for the development of new antimicrobial drugs against pathogenic relatives of C. glutamicum