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Proteolysis is essential for all living organisms to maintain the protein homeostasis and to adapt to changing environmental conditions. ClpP is the main protease in Bacillus subtilis, and forms complexes with different Clp ATPases. These complexes play crucial roles during heat stress, but also in sporulation or cell morphology. Especially enzymes of cell wall-, amino acid-, and nucleic acid biosynthesis are known substrates of the protease ClpP during glucose starvation. The aim of this study was to analyze the influence of a clpP mutation on the metabolism in different growth phases and to search for putative new ClpP substrates. Therefore, B. subtilis 168 cells and an isogenic ∆clpP mutant were cultivated in a chemical defined medium, and the metabolome was analyzed by a combination of 1H-NMR, HPLC-MS, and GC-MS. Additionally, the cell morphology was investigated by electron microscopy. The clpP mutant showed higher levels of most glycolytic metabolites, the intermediates of the citric acid cycle, amino acids, and peptidoglycan precursors when compared to the wild-type. A strong secretion of overflow metabolites could be detected in the exo-metabolome of the clpP mutant. Furthermore, a massive increase was observed for the teichoic acid metabolite CDP-glycerol in combination with a swelling of the cell wall. Our results show a recognizable correlation between the metabolome and the corresponding proteome data of B. subtilis clpP mutant. Moreover, our results suggest an influence of ClpP on Tag proteins that are responsible for teichoic acids biosynthesis.
Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.