Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

Abstract

Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx(DD) (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxΔC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.

Fig. 1.

BsYjbH mutants show similar activity to wild-type BsYjbH. (a) Effect of the wild-type and mutant on Spx-dependent expression of trxB–lacZ. The graph shows the IPTG induction time-course of β-galactosidase activity encoded by the trxB–lacZ fusion in yjbH mutant cells bearing the P_hyspank–BsyjbH (white) and mutants [coloured (see key)] or the P_hyspank promoter (black) in a yjbH-null mutant background. β-Galactosidase activity is expressed as Miller units. (b) Immunoblot analysis of Spx level using anti-Spx antiserum (Nakano et al., 2002). Cell samples were collected before IPTG induction and at 1 h after IPTG induction from the strains in (a).

Fig. 2.

GtYjbH mutants show similar activities to BsYjbH. (a) BsYjbH and GtYjbH affect Spx-dependent expression of trxB–lacZ. The graph shows the time-course of β-galactosidase activity encoded by the trxB–lacZ fusion in cells bearing the P_hyspank–BsyjbH (white) and GtyjbH (grey) or the P_hyspank promoter (black) in a yjbH-null mutant background. (b) Immunoblot analysis of Spx level before and after 1 mM diamide treatment at 0 (DI0), 10 (DI10), 25 (DI25) min time points. UI denotes uninduced. Cells were induced by IPTG for 30 min before diamide treatment. Anti-Spx antiserum, anti-BsYjbH antiserum and anti-His antibodies were used to detect Spx, BsYjbH and GtYjbH–His₆ levels. Control is purified Spx, BsYjbH–Strep and GtYjbH–His₆ protein.

Fig. 3.

GtYjbH enhanced Spx proteolysis mediated by ClpXP, which is ATP- and ClpX-dependent. (a, b) SDS-PAGE shows the effect of GtYjbH on ClpXP-catalysed proteolysis of Spx in vitro in a time-course experiment. Spx (8 µM), ClpX (3 µM) and ClpP (8 µM) with an ATP-generating system (creatine kinase) were incubated at 37 °C for the times (min) indicated in the absence (a) or presence (b) of GtYjbH (4 µM). (c) Plot of Spx band intensities against the reaction time in the experiments shown in the graph. The intensities of ClpP protein in each lane were used as internal controls (Zhang & Zuber, 2007). The Spx : ClpP ratio at 0 min was defined as 100 %. (d) Proteolysis assay under the same conditions as in (a, b) but in the presence of 0.16 µM GtYjbH.

Fig. 4.

GtYjbH recognizes the C terminus of Spx. Deletion of 12 amino acids at the Spx C terminus (SpxΔC) greatly diminished GtYjbH binding. (a) SDS-PAGE shows the effect of GtYjbH on ClpXP-catalysed proteolysis of SpxΔC in vitro in a time-course experiment. SpxΔC (4 µM), ClpX (3 µM) and ClpP (8 µM) with an ATP-generating system (creatine kinase) were incubated at 37 °C for the times (min) indicated in the absence (a) or presence (b) of GtYjbH (4 µM). (b) Far-Western blotting to detect Spx interaction with GtYjbH. Proteins that interact with SpxHA or SpxΔCHA were detected by anti-HA antibodies. RNA polymerase α subunit was used as a positive control for Spx interaction. (c, d) In vitro Ni-affinity interaction experiments to detect contact between GtYjbH–His₆ and Spx (c) or SpxΔC (d). M, marker; I, input; FT, flowthrough; W, last wash; E1–3, elution fractions 1–3.

Fig. 5.

Competition in vitro Ni-affinity interaction experiments show Spx outcompetes SpxΔC for GtYjbH binding. (a) Spx WT and SpxΔC proteins applied to a Ni affinity column. Column fractions separated by SDS-PAGE and stained with ‘Blue silver’ colloidal Coomassie G-250 protein. (b) Spx WT, SpxΔC and GtYjbH–His₆ reaction applied to Ni affinity column. Fractions separated by SDS-PAGE with staining as in (a). M, marker; I, input; FT, flowthrough; W, last wash; E1–3, elution fractions 1–3.

Fig. 6.

In vitro Ni-affinity interaction experiments to detect interaction between GtYjbH–His₆, Spx and ClpX, in the absence (a) or presence (b) of ClpP; between His₆–Spx and ClpX, in the absence or presence (c) of ClpP, all reactions contain ATP-γ-S; between GtYjbH–His₆, Spx^DD, ClpX and ClpP, in the presence or absence (d) of ATP-γ-S. Column fractions were applied to SDS-PAGE gels that were stained with ‘Blue silver’ colloidal Coomassie G-250 protein. M, marker; I, input; FT, flowthrough; W, last wash; E, elution.

Fig. 7.

A model for the mechanism of YjbH-enhanced proteolysis is proposed. In the absence of YjbH, the Spx C terminus contacts ClpX, but ClpXP proteolysis is slow. YjbH binds to the Spx C-terminal region, which may alter Spx folding, expose a structural element of Spx or eliminate Spx–ClpX contacts that might be inhibitory, thus accelerating Spx proteolysis.