The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.     

Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.     

The ATPase ClpX is conditionally involved in the morphological differentiation of <Emphasis Type="Italic"> Streptomyces lividans</Emphasis>

ATP-dependent proteases of the ClpP type are widespread in eubacteria. These proteolytic complexes are composed of a proteolytic subunit and an ATPase subunit. They are involved in the degradation of denatured proteins, but also play a role in specific regulatory pathways. In Streptomyces lividans strains which lack the proteolytic subunit ClpP1, cell cycle progression has been shown to be blocked at early stages of growth. In this study, we examined the role of the ATPase subunit ClpX, a possible partner of the products of the clpP1 operon. A clpX mutant was obtained and it was shown that its growth was impaired only on acidic medium. Thus, the clpX phenotype differs from the clpP1 phenotype, indicating that these two components have only partially overlapping roles. We also analyzed the expression of clpX. Although clpX expression is increased under heat-shock conditions in many bacteria, we found that this is not the case in S. lividans.     

Global role for ClpP-containing proteases in stationary-phase adaptation of Escherichia coli

To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.     

Essentiality of clpX,but not clpP,clpL, clpC,or clpE,in Streptococcus pneumoniae R6

We show by using a regulated promoter that clpX of Streptococcus pneumoniae R6 is essential, whereas clpP, clpL, clpC, and clpE can be disrupted. The essentiality of clpX was initially missed because of duplication and rearrangement in the region of the chromosome containing clpX. Depletion of ClpX resulted in a rapid loss of viability without overt changes in cell morphology. Essentiality of clpX, but not clpP, has not been reported previously.     

Sodium dodecyl sulfate hypersensitivity of clpP and clpB mutants of Escherichia coli

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.     

The interplay of ClpXP with the cell division machinery in Escherichia coli

ClpXP is a two-component protease composed of ClpX, an ATP-dependent chaperone that recognizes and unfolds specific substrates, and ClpP, a serine protease. One ClpXP substrate in Escherichia coli is FtsZ, which is essential for cell division. FtsZ polymerizes and forms the FtsZ ring at midcell, where division occurs. To investigate the role of ClpXP in cell division, we examined the effects of clpX and clpP deletions in several strains that are defective for cell division. Together, our results suggested that ClpXP modulates cell division through degradation of FtsZ and possibly other cell division components that function downstream of FtsZ ring assembly. In the ftsZ84 strain, which is temperature sensitive for filamentation due to a mutation in ftsZ, we observed that deletion of clpX or clpP suppresses filamentation and reduces FtsZ84 degradation. These results are consistent with ClpXP playing a role in cell division by modulating the level of FtsZ through degradation. In another division-defective strain, ΔminC, the additional deletion of clpX or clpP delays cell division and exacerbates filamentation. Our results demonstrate that ClpXP modulates division in cells lacking MinC by a mechanism that requires ATP-dependent degradation. However, antibiotic chase experiments in vivo indicate that FtsZ degradation is slower in the ΔminC strain than in the wild type, suggesting there may be another cell division component degraded by ClpXP. Taken together these studies suggest that ClpXP may degrade multiple cell division proteins, thereby modulating the precise balance of the components required for division.