title: Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis.
abstract: The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.
authors.affiliation: Applied Microbiology Division, National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Thi-Huyen
authors.initials: TH
authors.lastname: Do
authors.forename: Yuki
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Naoki
authors.initials: N
authors.lastname: Abe
authors.forename: Jun
authors.initials: J
authors.lastname: Kaneko
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Itoh
authors.forename: Keitarou
authors.initials: K
authors.lastname: Kimura
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D055786:Gene Knockout Techniques; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D015534:Trans-Activators / Q000172:deficiency*
chemical_list: D001426:Bacterial Proteins; C091639:DegQ protein, Bacillus sp.; C066496:DegS protein, Bacteria; C066497:DegU protein, Bacteria; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D010744:Phosphoric Monoester Hydrolases