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 SpxDD (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.
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