Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis

Thi Huyen Do, Yuki Suzuki, Naoki Abe, Jun Kaneko, Yoshifumi Itoh, Keitarou Kimura

    Research output: Contribution to journalArticlepeer-review

    28 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)8249-8258
    Number of pages10
    JournalApplied and environmental microbiology
    Volume77
    Issue number23
    DOIs
    Publication statusPublished - 2011 Dec

    ASJC Scopus subject areas

    • Biotechnology
    • Food Science
    • Applied Microbiology and Biotechnology
    • Ecology

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