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Projekt Druckansicht

Aufdeckung des molekularen Mechanismus der Dekryptifizierung des gudB-Gens in dem Gram-positiven Modellbakterium Bacillus subtilis

Fachliche Zuordnung Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Allgemeine Genetik und funktionelle Genomforschung
Förderung Förderung von 2012 bis 2020
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 223782191
 
Erstellungsjahr 2020

Zusammenfassung der Projektergebnisse

The Gram-positive model bacterium Bacillus subtilis exclusively relies on the glutamine synthetase (GS) and the glutamate synthase (GltAB) for de novo synthesis of glutamate. The glutamate dehydrogenase (GDH) RocG is strictly catabolically active and allows the bacteria to utilize glutamate as a source of carbon and nitrogen. During growth with the preferred carbon source glucose, the LysR-type transcription factor activates the transcription of the gltAB glutamate synthase genes to meet the cellular need for glutamate. By contrast, in the presence of glutamate, the GDH RocG degrades glutamate and simultaneously inhibits the DNA-binding activity of GltC, thereby preventing glutamate biosynthesis. The carbon and nitrogen sourcedependent control of glutamate biosynthesis allows the efficient utilization of the available nutrients. Previous experimental evidence suggested that the DNA-binding activity of GltC is stimulated and inhibited by 2-oxoglutarate and glutamate, respectively. However, the metabolite-dependent control of GltC activity does not seem to be relevant in vivo because the GDH RocG is the major factor controlling glutamate biosynthesis in B. subtilis. Laboratory strains of B. subtilis also produce a catalytically inactive GDH, which is encoded by the cryptic gudBCR gene. The gene encodes a non-functional enzyme because it contains a perfect 18 bp-long tandem repeat, which causes a duplication of three amino acids in the active center of the GDH GudB. Previously, it has been shown that a B. subtilis rocG mutant strain rapidly accumulates mutations in the gudBCR gene and the suppressor mutants always express the gudB gene lacking one unit of the 18 bp-long tandem repeat. The gudB gene encodes the functional GDH GudB1, which is also capable of controlling glutamate biosynthesis via the interaction with GltC. The research proposal was aimed at elucidating the molecular mechanism of tandem repeat mutagenesis in the bacterium B. subtilis. We could not demonstrate that factors involved DNA repair are involved in tandem repeat mutagenesis. However, the transcription machinery strongly decreases the integrity of tandem repeats in B. subtilis. As part of this project, a genetic system was developed that allowed us to visualize tandem repeat mutagenesis in single cells. This system will be helpful to study the effects of the selective pressure and of the transcription machinery on the frequency of tandem repeat mutagenesis. Since there was no obvious relationship between DNA repair factors and tandem repeat mutagenesis, we also focused on elucidating the molecular mechanism underlying the control of glutamate biosynthesis in B. subtilis. We could demonstrate that the DNA binding transcription factor GltC can act as an activator and as a repressor of the gltAB glutamate synthase genes. Moreover, experimental evidence suggests that the GDHs RocG and GudB1 convert the transcriptional activator GltC into a repressor of the gltAB genes in a glutamatedependent manner. Thus, we have discovered a novel mode of gene regulation in B. subtilis.

Projektbezogene Publikationen (Auswahl)

  • 2017. Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant. Environ. Microbiol. Rep. 9: 279-289
    Dormeyer, M., Lübke, A. L., Müller, P., Lentes, S., Reuß, D. R., Thürmer, A., Stülke, J., Daniel, R., Brantl, S. & Commichau, F. M.
    (Siehe online unter https://doi.org/10.1111/1758-2229.12531)
  • 2018. Changes in DNA topology affect the global transcription landscape and allow rapid growth of a Bacillus subtilis strain lacking carbon catabolite repression. Metab. Eng. 45: 171-179
    Reuß, D. R., Rath, H., Thürmer, A., Benda, M., Daniel, R., Völker, U., Mäder, U., Commichau, F. M. & Stülke, J.
    (Siehe online unter https://doi.org/10.1016/j.ymben.2017.12.004)
  • 2018. Selective pressure for biofilm formation in Bacillus subtilis: differential effect of mutations in the master regulator SinR on bistability. mBio. 9: pii: e01464-18
    Kampf, J., Gerwig, J., Kruse, K., Cleverley, R., Dormeyer, M., Grünberger, A., Kohlheyer, D., Commichau, F. M., Lewis, R. J. & Stülke, J.
    (Siehe online unter https://doi.org/10.1128/mBio.01464-18)
  • 2018. Visualization of tandem repeat mutagenesis in Bacillus subtilis. DNA Repair (Amst). 63C: 10-15
    Dormeyer, M., Lentes, S., Ballin, P., Wilkens, M., Klumpp, S., Kohlheyer, D., Stannek, L., Grünberger, A. & Commichau, F. M.
    (Siehe online unter https://doi.org/10.1016/j.dnarep.2018.01.006)
  • 2019. Variants of the Bacillus subtilis LysR-type regulator GltC with altered activator and repressor function. Front. Microbiol. 10: 2321
    Dormeyer, M., Lentes, S., Richts, B., Heermann, R., Ischebeck, T. & Commichau, F. M.
    (Siehe online unter https://doi.org/10.3389/fmicb.2019.02321)
 
 

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