ClpXP is a protease complex that plays important roles in protein quality control and cell cycle regulation, but the functions of multiple ClpXs and multiple ClpPs in M. xanthus remain unknown. The genome of Myxococcus xanthus DK1622 contains two clpPs and three clpXs. The clpP1 and clpX1 genes are cotranscribed and are both essential, while the other copies are isolated in the genome and are deletable. The deletion of clpX2 caused the mutant to be deficient in fruiting body development, while the clpX3 gene is involved in resistance to thermal stress. Both ClpPs possess catalytic active sites, but only ClpP1 shows in vitro peptidase activity on the typical substrate Suc-LY-AMC. All of these clpP and clpX genes exhibit strong transcriptional upregulation in the stationary phase, and the transcription of the three clpX genes appears to be coordinated. Our results demonstrated that multiple ClpPs and multiple ClpXs are functionally divergent and may assist in the environmental adaptation and functional diversification of M. xanthus.IMPORTANCEClpXP is an important protease complex of bacteria and is involved in various physiological processes. Myxococcus xanthus DK1622 possesses two ClpPs and three ClpXs with unclear functions. We investigated the functions of these genes and demonstrated the essential roles of clpP1 and clpX1. Only ClpP1 has in vitro peptidase activity on Suc-LY-AMC, and the isolated clpX copies participate in distinct cellular processes. All of these genes exhibited significant transcriptional upregulation in the stationary phase. Divergent functions appear in multiple ClpPs and multiple ClpXs in M. xanthus DK1622.

In cells, proteins are synthesized, function, and degraded (dead). Protein synthesis (spring) is important for the life of proteins. However, how proteins die is equally important for organisms. Proteases are secreted from cells and used as nutrients to break down external proteins. Proteases degrade unwanted and harmful cellular proteins. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for cellular protein degradation. Prokaryotes, such as bacteria, have similar protein degradation systems. In this review, we describe the structure and function of the ClpXP complex in the degradation system, which is an ATP-dependent protease in bacterial cells, with a particular focus on ClpP.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome; however, the exact role of molecular chaperones in this critical process remains unclear. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of bacterial cell division by showing the growth defect of a Staphylococcus aureus clpX mutant is rescued by a spontaneously acquired G325V substitution in the ATP-binding domain of the essential FtsA cell division protein. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with Escherichia coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division.

Mitochondrial fatty acid oxidation (β-oxidation) is an essential metabolic process for energy production in eukaryotic cells, but the regulatory mechanisms of this pathway are largely unknown. In the present study, we found that several enzymes involved in β-oxidation are associated with CLPX, the AAA+ unfoldase that is a component of the mitochondrial matrix protease ClpXP. The suppression of CLPX expression increased β-oxidation activity in the HepG2 cell line and in primary human hepatocytes without glucagon treatment. However, the protein levels of enzymes involved in β-oxidation did not significantly increase in CLPX-deleted HepG2 cells (CLPX-KO cells). Coimmunoprecipitation experiments revealed that the protein level in the immunoprecipitates of each antibody changed after the treatment of WT cells with glucagon, and a part of these changes was also observed in the comparison of WT and CLPX-KO cells without glucagon treatment. Although the exogenous expression of WT or ATP-hydrolysis mutant CLPX suppressed β-oxidation activity in CLPX-KO cells, glucagon treatment induced β-oxidation activity only in CLPX-KO cells expressing WT CLPX. These results suggest that the dissociation of CLPX from its target proteins is essential for the induction of β-oxidation in HepG2 cells. Moreover, specific phosphorylation of AMP-activated protein kinase and a decrease in the expression of acetyl-CoA carboxylase 2 were observed in CLPX-KO cells, suggesting that CLPX might participate in the regulation of the cytosolic signaling pathway for β-oxidation. The mechanism for AMP-activated protein kinase phosphorylation remains elusive; however, our results uncovered the hitherto unknown role of CLPX in mitochondrial β-oxidation in human liver cells.

The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

Protein homeostasis is tightly regulated by protein quality control systems such as chaperones and proteases. In cyanobacteria, the ClpXP proteolytic complex is regarded as a representative proteolytic system and consists of a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. However, the functions and molecular mechanisms of ClpX in cyanobacteria remain unclear. This study aimed to decipher the unique contributions and regulatory networks of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We showed that the interruption of clpX led to slower growth, decreased high light tolerance, and impaired photosynthetic cyclic electron transfer. A quantitative proteomic strategy was employed to globally identify ClpX-regulated proteins in Synechocystis cells. In total, we identified 172 differentially expressed proteins (DEPs) upon the interruption of clpX. Functional analysis revealed that these DEPs are involved in diverse biological processes, including glycolysis, nitrogen assimilation, photosynthetic electron transport, ATP-binding cassette (ABC) transporters, and two-component signal transduction. The expression of 24 DEPs was confirmed by parallel reaction monitoring (PRM) analysis. In particular, many hypothetical or unknown proteins were found to be regulated by ClpX, providing new candidates for future functional studies on ClpX. Together, our study provides a comprehensive ClpX-regulated protein network, and the results serve as an important resource for understanding protein quality control systems in cyanobacteria.

Despite having a highly reduced genome, Chlamydia trachomatis undergoes a complex developmental cycle in which the bacteria differentiate between the following two functionally and morphologically distinct forms: the infectious, nonreplicative elementary body (EB) and the noninfectious, replicative reticulate body (RB). The transitions between EBs and RBs are not mediated by division events that redistribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require bulk protein turnover. One system for targeted protein degradation is the trans-translation system for ribosomal rescue, where polypeptides stalled during translation are marked with an SsrA tag encoded by a hybrid tRNA-mRNA, tmRNA. ClpX recognizes the SsrA tag, leading to ClpXP-mediated degradation. We hypothesize that ClpX functions in chlamydial differentiation through targeted protein degradation. We found that mutation of a key residue (R230A) within the specific motif in ClpX associated with the recognition of SsrA-tagged substrates resulted in abrogated secondary differentiation while not reducing chlamydial replication or developmental cycle progression as measured by transcripts. Furthermore, inhibition of trans-translation through chemical and targeted genetic approaches also impeded chlamydial development. Knockdown of tmRNA and subsequent complementation with an allele mutated in the SsrA tag closely phenocopied the overexpression of ClpXR230A, thus suggesting that ClpX recognition of SsrA-tagged substrates plays a critical function in secondary differentiation. Taken together, these data provide mechanistic insight into the requirements for transitions between chlamydial developmental forms. IMPORTANCE Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and preventable infectious blindness. This unique organism undergoes developmental transitions between infectious, nondividing forms and noninfectious, dividing forms. Therefore, the chlamydial developmental cycle is an attractive target for Chlamydia-specific antibiotics, which would minimize effects of broad-spectrum antibiotics on the spread of antibiotic resistance in other organisms. However, the lack of knowledge about chlamydial development on a molecular level impedes the identification of specific, druggable targets. This work describes a mechanism through which both the fundamental processes of trans-translation and proteomic turnover by ClpXP contribute to chlamydial differentiation, a critical facet of chlamydial growth and survival. Given the almost universal presence of trans-translation and ClpX in eubacteria, this mechanism may be conserved in developmental cycles of other bacterial species. Additionally, this study expands the fields of trans-translation and Clp proteases by emphasizing the functional diversity of these systems throughout bacterial evolution.

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is easy to alter. In vitro, wild-type ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that the rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states.

Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied.