Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU‐P by ClpCP

Abstract

SummaryThe response regulator DegU and its cognate kinase DegS constitute a two-component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU-lacZ fusions, we showed that phosphorylated DegU (DegU-P) binds to two direct repeats (DR1 and DR2) of the consensus DegU-binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATPdependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild-type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU-P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU-P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N-terminal region of DegU is important for proteolysis.

SCF KMD controls cytokinin signaling by regulating the degradation of type-B response regulators

“…In addition, proteasomedependent degradation of ARR2, a type-B ARR, is enhanced by cytokinin-induced phosphorylation of the conserved Asp residue in the receiver domain (17). The two-component signaling system is evolutionarily ancient (36), and our study points to how plants, like bacteria, use degradation of response regulators as a pivotal mechanism for transcriptional regulation (37,38). Plants, unlike bacteria, accomplish this by using the eukaryotic-specific ubiquitinproteasome system.…”

The Bacillus subtilis Response Regulator Gene degU Is Positively Regulated by CcpA and by Catabolite-Repressed Synthesis of ClpC

“…We used polyclonal anti-DegU (38), antiClpP (39), and anti-ClpC (40) antibodies for analyses. Quantification of band intensities was described previously (26).…”

“…For EMSA, the indicated amounts of His-CcpA were incubated with 5 nM DNA probe in 12 l of 0.5ϫ TEDG buffer containing 20 mM KCl, 1 mM MgCl 2 , and an appropriate amount of poly(dI-dC) as a nonspecific competitor (GE Healthcare, United Kingdom) for 15 min at 37°C. After addition of 2 l of loading buffer (26), the samples were separated in polyacrylamide gels containing the indicated concentrations of acrylamide. Electrophoresis was performed in 0.25ϫ Tris-borate-EDTA (TBE) buffer at 4°C.…”

“…These observations indicate that there is some nutrient-dependent regulation of degU. Furthermore, the AAA ϩ protease ClpCP specifically degrades the phosphorylated form of DegU (DegU-P), leading to modulation of the DegU autoactivation loop (26,30). DegU is also regulated by protein-protein interactions, including the RapG-PhrG system.…”

“…In its high-level phosphorylation state, DegU activates degU itself and induces many target genes encoding extracellular degrading enzymes (20)(21)(22)(23)(24)(25). Thus, degU is activated by an autoregulatory loop through the P3 promoter (26,27). In addition, high levels of DegU-P result in repression of motility (25,28).…”