Mutations in B. subtilis ClpX residues predicted to be required for interaction with canonical substrates disrupt ClpXP degradation of Spx. (A) Alignment of B. subtilis and E. coli ClpX polypeptides. Amino acid identity is indicated by black boxes and amino acid similarity by gray boxes. Bars indicate Walker A, pore loop, and Walker B boxes, respectively. Numbers refer to the B. subtilis ClpX sequence. Arrows indicate residues that were changed to alanine for this study. (B) Quantitative immunoblot of lysates from mid-exponential-phase cultures grown in the absence (−) or presence (+) of xylose. In strains encoding clpX under the control of both its native promoter and the exogenous P_xyl promoter, ClpX levels were ∼2-fold lower than those in the parent strain in both the mutant and congenic wild-type backgrounds in the absence of xylose. Xylose-induced expression from the P_xyl promoter raised the intracellular concentration of ClpX to wild-type levels in these strains. Lysates were blotted with anti-ClpX sera (top) or anti-FtsZ sera (bottom). ClpX appears as a doublet on immunoblots. Mutations in clpX do not affect FtsZ levels. (C) Colonies stabbed onto tryptone broth swarm plates in the presence and absence of xylose. Note that only the congenic wild-type strain is motile, indicating appropriate turnover of Spx. Minor differences in swarm size in the congenic wild-type strain are most likely the result of experimental variation and are not a reflection of changes in ClpX levels. (D) Immunoblot of lysates from mid-exponential cultures probed with antisera against the ClpX target Spx or with anti-FtsZ sera. Note the increased levels of Spx in the four clpX mutant strains, confirming a defect in the ClpX-Spx interaction. FtsZ serves as a loading control.

Mutations in residues required for ClpXP-mediated proteolysis have a negligible effect on the ability of ClpX to inhibit FtsZ assembly. (A) Suppression of the lethality associated with >12-fold overexpression of the MinCD division inhibitor. Both the Walker B (E182A) and pore loop (Y150A) mutants are sensitive to MinCD overexpression in the presence or absence of xylose, indicating that they are wild type with regard to inhibiting FtsZ assembly. In contrast, the predicted ClpX Walker A mutant (K122A) is sensitive to high levels of MinCD only when expressed at wild-type levels in the presence of xylose. A twofold reduction in the ClpX(K122A) concentration by growth in the absence of xylose results in an increase in resistance to MinCD overexpression. Error bars indicate standard deviations from at least three separate experiments. (B) Representative traces of 90°-angle light-scattering reactions containing 3 μM FtsZ assembled alone or in the presence of wild-type ClpX (top) or a mutant that is defective in ATP hydrolysis, ClpX(E182A). (C) Histogram of FtsZ assembly reactions conducted in the presence of the wild-type or mutant ClpX protein. Each bar represents at least three replicate experiments. Error bars indicate standard deviations from three separate experiments.