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.     

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.     

An essential protease involved in bacterial cell-cycle control.

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.     

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.     

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.     

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.     

ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems

Efficient acquisition of genes that encode a restriction and modification (R–M) system with specificities different from any already present in the recipient bacterium requires the sequential production of the new modification enzyme followed by the restriction activity in order that the chromosome of the recipient bacterium is protected against attack by the restriction endonuclease. We show that ClpX and ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the genes encoding Eco KI and Eco AI, representatives of two families of type I R–M systems, thus implicating ClpXP in the modulation of restriction activity. Loss of ClpX imposed a bigger barrier than loss of ClpP, consistent with a dual role for ClpX, possibly as a chaperone and as a component of the ClpXP protease. Transmission of genes specifying Eco KI was more dependent on ClpX and ClpP than transmission of the genes for Eco AI. Sensitivity to absence of the protease was also influenced by the mode of gene transfer; conjugative transfer and transformation were more dependent on ClpXP than transduction. In the absence of either ClpX or ClpP transfer of the Eco KI genes by P1-mediated transduction was impaired, transfer of the Eco AI genes was not.     

Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase

The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.     

Precise amounts of a novel member of a phosphotransferase superfamily are essential for growth and normal morphology in Caulobacter crescentus

The Caulobacter crescentus chromosomal clp locus contains the genes encoding the components of ClpXP, a multisubunit protease required for cell cycle progression in this organism. Here, we report the identification and characterization of cicA, a gene located between the clpX and clpP genes on the Caulobacter chromosome. cicA is a novel morphogene in C. crescentus and, like clpX and clpP, is essential for growth. A conditional cicA mutant stopped growth, but retained viability under restrictive conditions. In contrast, an increased concentration of CicA led to an immediate loss of the normal rod shape, an almost 10-fold increase of the cell's volume and a cell division block. In parallel with this drastic morphological change, cells rapidly lost viability. Primary sequence analysis suggested that the cicA gene encodes a member of a large superfamily of phosphotransferases, that include phosphoserine phosphatases, the ATPase domain of P-type ATPases and receiver domains of response regulators. Four conserved motifs of this protein family that have been implicated in the catalysis of phosphotransfer reactions were investigated by site-directed mutagenesis and were found to be critical for in vivo function of CicA. Based on our observations, we postulate that CicA is involved in essential phosphotransferase reactions in Caulobacter and that increased activity of CicA has a deleterious effect on cell wall biosynthesis, morphogenesis and cell division.     

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.     

Communication between ClpX and ClpP during substrate processing and degradation

In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.     

ClpXP, an ATP-powered unfolding and protein-degradation machine

ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies.     

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.     

Involvement of Clp protease activity in modulating the Bacillus subtilissigmaw stress response

The induction of Bacillus subtilis genes controlled by the extracytoplasmic function alternative sigma factor sigmaW is strongly impaired in a strain deleted for the ClpP peptidase gene and in a double knockout of the ClpX and ClpE ATPase genes. Truncated soluble forms of the sigmaW anti-sigma factor RsiW are stabilized in a clpP minus strain as revealed by the green fluorescent reporter protein fused to the N-terminus of RsiW and by pulse-chase experiments. Conserved alanine residues are present in the transmembrane region of RsiW, and mutations in these positions abolish induction of sigmaW-controlled genes. Following alkaline shock, a truncated cytoplasmic form of RsiW is detectable in a strain expressing a triple alanine mutant allele of rsiW. These data point to a mechanism where the trans-membrane segment of RsiW contains a cryptic proteolytic tag that is uncovered as a result of intramembrane proteolysis of RsiW by RasP (YluC). After RasP-clipped RsiW is detached from the membrane, this proteolytic tag becomes crucial for the complete degradation of RsiW by cytoplasmic proteases and the release of sigmaW. ClpXP plays a major role in this third proteolytic step of stress-induced degradation of RsiW. Overexpression of SsrA-tagged green fluorescent protein as a ClpXP substrate protein reduces alkali induction of a sigmaW-controlled gene by a factor of about three, indicating that a titration mechanism is able to tune the sigmaW-mediated stress response to the cellular state.     

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.