Maintenance of glutamate homeostasis in Bacillus subtilis by complex regulatory systems and genomic adaptation

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.