Control of glutamate homeostasis in the Gram-positive model organism Bacillus subtilis
von Lorena Stannek
Datum der mündl. Prüfung:2015-04-28
Erschienen:2015-10-22
Betreuer:Dr. Fabian Moritz Commichau
Gutachter:Dr. Fabian Moritz Commichau
Gutachter:Prof. Dr. Jörg Stülke
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-5321
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Name:Lorena Stannek Dissertation.pdf
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Zusammenfassung
Englisch
Glutamate is a central metabolite in any living organism because it is the major amino group donor for nitrogen containing compounds. To ensure a constant glutamate supply, Bacillus subtilis tightly regulates glutamate metabolism in dependence of the available carbon and nitrogen sources. The only glutamate synthesizing reaction in B. subtilis is the reductive amination of 2-oxoglutarate by the glutamate synthase GOGAT. The glutamate dehydrogenases (GDHs) of B. subtilis are strictly devoted to glutamate degradation. The genome of the B. subtilis laboratory strain 168 contains the rocG and gudBCR genes, encoding the active GDH RocG and the highly instable and inactive GDH GudBCR, respectively. While transcription of the rocG gene is tightly regulated, the gudBCR gene is constitutively expressed. Upon deletion of the gene encoding RocG, B. subtilis forms suppressor mutants on complex medium. The suppressor mutants synthesize a stable and active GudB+ enzyme, thereby keeping the glutamate metabolism in balance. The aim of this work was to shed more light on the complex regulation of glutamate metabolism and to dissect the role of the GDHs within B. subtilis. It has been unclear, why the gudBCR gene encoding the inactive GDH GudBCR is stably inherited over many generations in the B. subtilis laboratory strain 168. Here, for the first time a plausible explanation for the stable inheritance of the gudBCR allele under laboratory growth conditions is provided. Compared to a strain synthesizing the active GDHs RocG and GudB+, the laboratory strain producing only RocG has a selective growth advantage when glutamate is scarce. By contrast, with excess of exogenous glutamate the strain expressing the GDHs RocG and GudB+ rapidly outcompetes a strain synthesizing only RocG. Thus, the level of GDH activity strongly influences fitness of the bacteria depending on the availability of glutamate. In the present work, a mechanism for the degradation of the inactive GDH GudBCR is also proposed. It could be demonstrated, that the arginine kinase McsB and the cognate phosphatase YwlE affect the stability of the GudBCR protein. Furthermore, interaction-studies suggest that the ClpCP protease complex is involved in the degradation of the GudBCR protein. Finally, in this work it could be demonstrated that like the GDH RocG also GudB+ acts as a trigger enzyme that controls the DNA-binding activity of GltC, which is the transcriptional activator of the GOGAT-encoding gltAB genes. Moreover, the essential role of glutamate for the GDH-dependent control of GltC was demonstrated. Only when the internal glutamate concentrations are sufficiently high, the GDH inhibits the DNA binding activity of GltC. Thus, the GDHs function as sensors of the cellular glutamate pool and regulate the glutamate synthesis through interaction with GltC. The finding of the synergistic control of glutamate biosynthesis by glutamate and the GDHs deepens our understanding of the regulation of glutamate metabolism in B. subtilis.
Keywords: Bacillus subtilis; glutamate; microbiology; subtilis; amino acids; cocultivation; proteolysis; glutamate dehydrogenase; trigger enzyme; bacteria; GudB