dc.contributor.advisor | Commichau, Fabian Moritz Dr. | |
dc.contributor.author | Dormeyer, Miriam | |
dc.date.accessioned | 2017-10-16T09:28:04Z | |
dc.date.available | 2017-10-16T09:28:04Z | |
dc.date.issued | 2017-10-16 | |
dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0023-3F32-8 | |
dc.identifier.uri | http://dx.doi.org/10.53846/goediss-6530 | |
dc.language.iso | eng | de |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
dc.subject.ddc | 570 | de |
dc.title | Maintenance of glutamate homeostasis in Bacillus subtilis by complex regulatory systems and genomic adaptation | de |
dc.type | doctoralThesis | de |
dc.contributor.referee | Stülke, Jörg Prof. Dr. | |
dc.date.examination | 2017-10-12 | |
dc.description.abstracteng | The Gram-positive model organism Bacillus
subtilis lives in the soil and must cope with a
constantly changing environment. Glutamate
plays an important role in cellular metabolism,
because it is the major amino group donor and it
serves as a precursor for proline, which is an
osmoprotectant in B. subtilis. The reactions
involved in anabolism and catabolism of
glutamate represent an important metabolic
node, linking carbon to nitrogen metabolism.
The glutamine synthetase (GS) and the
glutamate synthase (GOGAT) forming the GSGOGAT cycle, are responsible for nitrogen
assimilation in B. subtilis. The GS uses ATP to
produce glutamine from ammonium and
glutamate and the GOGAT catalyzes the
conversion of glutamine and α-ketoglutarate to
two molecules of glutamate. The glutamate
dehydrogenase (GDH) is strictly catabolically
active and oxidizes glutamate to ammonium and
α-ketoglutarate. To ensure a constantly high
level of glutamate, the anabolic and catabolic
reactions involved in glutamate metabolism have
to be tightly controlled by signals derived from
nitrogen and carbon metabolism. Perturbation
of glutamate homeostasis causes a severe
growth defect of B. subtilis. To adjust glutamate
synthesis to the cellular demand for glutamate,
expression of the GOGAT encoding gltAB genes
is strictly controlled. This is achieved by
controlling the DNA-binding activity of the
transcription factor GltC, which regulates
expression of the gltAB genes. It was found in
vivo that the GDH RocG in B. subtilis can bind GltC
in the presence of glutamate and thereby
prevents the expression of the gltAB genes and
the emergence of a futile cycle of glutamate
synthesis and degradation. In vitro, it was found
that GltC, which prevents the RNAP from
transcribing the gltAB genes acts as a glutamatedependent repressor. In this work, it is shown
that RocG triggers the repressor function of GltC
resulting in the formation of a RocG-GltC
complex that binds to the promoter of the gltAB
genes. This model combines the two existing
models for the regulation of the gltAB genes to
one consistent model. The disturbance of this
highly complex regulation results in a severe
growth defect. For instance, a RocG deficient
strain cannot degrade glutamate, resulting in the
accumulation of glutamate. The accumulation of
glutamate is prevented in rapidly emerging
suppressor mutants (SM) that have mutated the
gudBCR gene. In the B. subtilis laboratory strain
168, the gudBCR gene harbors a tandem repeat
(TR) and encodes for a second inactive GDH. The
excision of one TR unit leads to the activation of
the gudB gene encoding the active GDH GudB
that can fully replace RocG. In this work, the
influence of several factors on the TR
mutagenesis of the gudB gene is investigated. In
contrast to a RocG deficient strain, a GltC
deficient strain cannot produce the GOGAT and
consequently it does not synthesize glutamate.
In this work, a selection and screening system is
used to show that several classes of mutations
can compensate for glutamate auxotrophy.
Class I mutants harbored promoter-up
mutations in the promoter of the gltAB genes. In
class II mutants the gltR gene acquired a single
mutation and the resulting GltR24 protein
replaces GltC. The majority of SMs were class III
mutants, harboring multiple copies of the gltAB
genes to increase the cellular amount of the
GOGAT.
To conclude, a genetic approach was employed
to generate a novel and consistent model
describing the control of glutamate biosynthesis
in B. subtilis. This work also revealed that
B. subtilis mutants with defects in glutamate
metabolism flexibly respond to perturbation of
glutamate homeostasis at the level of the
genome. | de |
dc.contributor.coReferee | Gatz, Christiane Prof. Dr. | |
dc.contributor.thirdReferee | Pöggeler, Stefanie Prof. Dr. | |
dc.contributor.thirdReferee | Konrad, Manfred Dr. | |
dc.contributor.thirdReferee | Klumpp, Stefan Prof. Dr. | |
dc.subject.eng | Evolution | de |
dc.subject.eng | Bacillus subtilis | de |
dc.subject.eng | Glutamate | de |
dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0023-3F32-8-1 | |
dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
dc.subject.gokfull | Biologie (PPN619462639) | de |
dc.identifier.ppn | 1002330580 | |