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 (␥PGA). To elucidate the role of DegQ in ␥PGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored ␥PGA 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.Extracellular poly-gamma-glutamate (␥PGA) is a glutamate polymer linked through a gamma-peptide bond produced outside the cells by bacilli, including Bacillus subtilis, B. licheniformis, and B. anthracis, and Staphylococcus epidermidis (4). It acts as a nutrient reservoir to prevent starvation in the stationary phase and as a barrier against bacteriophage or phagocytotic attack by the host immune system (4, 12, 14, 16). Microbial functions that are not essential for the life cycle under laboratory conditions but that are required for survival in the natural environment are sometimes lost in domesticated laboratory strains. The loss of the ability to synthesize ␥PGA in a laboratory strain of B. subtilis is an example of such an event, and a mutation in degQ is involved in it (30).degQ function was characterized mainly through studies of the ␥PGA-negative laboratory lineage B. subtilis 168. degQ encodes a small peptide of 46 amino acids, and it is a pleiotropic regulator of degradation enzymes, including alkaline protease, ␣-amylase, and levansucrase (1). The expression of degQ is dependent on comA, the global transcriptional regulator in the stationary phase, and is strongly induced under dense-cell conditions (18). The regulation of these exoenzymes by degQ requires the presence of the degS-degU operon that comprises a two-component system (17, 18). The sensor histidine kinase DegS has four activities: it acts as a self-kinase (autophosphorylation of His189), a self-phosphatase (autodephosphorylation), a phosphotransferase (phosphorylation of Asp56 of DegU), and a phosphatase (dephosphorylation of DegU) (5,6,15,20,31). Exoenzyme expression is controlled directly by DegU, a DNA-binding protein, and DegU phosphorylation by DegS (DegU-P i ) enhances it (25,29,34). A recent study reported that DegQ stimulates phosphate transfer from phospho...

Defects in the Flagellar Motor Increase Synthesis of Poly-γ-Glutamate in Bacillus subtilis

“…5). Consistent with previous reports, mutation of the DegS/DegU two-component systems abolished mucoidy in a motA background and reduced pgs expression to background levels (85,87,91). Transcription of pgs was similarly reduced when a motA background was also mutated for the Com quorum-sensing system, CodY, a nutrition-responsive transcription factor, or Mfd, a transcription-coupled DNA repair protein (62,71,89).…”

“…3B). Only three of the mutants did not regain mucoidy in the presence of IPTG: pgsB, which is required for PGA synthesis, and degS and degU, which encode the DegS and DegU two-component system through which DegQ is thought to act (85,90,91). We conclude that DegS and DegU are likely the most downstream regulators in pgs operon expression, as increased activity of the two-component system can override the other mutants defective in PGA production.…”

“…DegQ is a ComPA-regulated protein that enhances the phosphorylation of DegU by DegS (85,89,90,91). To test epistatic relationships to DegU, the gene encoding DegQ, degQ, was cloned downstream of the IPTG-inducible P hyspank promoter integrated at the ectopic amyE site in the mutant backgrounds that reduced production of PGA (amyE::P hyspank -degQ).…”

Improvement of Bacillus subtilis for poly‐γ‐glutamic acid production by genome shuffling

“…The mRNA level of pgs B in F3‐178 was 18.8‐fold higher than in GXA‐28, supporting the key role of pgs B in γ‐PGA production in F3‐178. More studies are needed to examine the level of other genes participated in γ‐PGA production, such as pgs E and deg Q (Do et al ., ; Yamashiro et al ., ).…”

Biocontrol of Fusarium wilt of banana: Key influence factors and strategies

“…It is a biodegradable, water-soluble amino acid polymer with a molecular weight ranging from about 10,000 up to 2 millions, generated by the microbial fermentation in nature . It is normally composed of about 5,000 glutamic acids molecules or mono units, with a free carboxyl group on the α-carbon atom of each repeating unit (Do et al, 2011).…”