Regulated Proteolysis in Bacteria.

Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.

Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis.

The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.

Structure and function of ClpXP, a AAA+ proteolytic machine powered by probabilistic ATP hydrolysis.

ClpXP is an archetypical AAA+ protease, consisting of ClpX and ClpP. ClpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas ClpP is a self-compartmentalized peptidase. ClpXP is currently the only AAA+ protease for which high-resolution structures exist, the molecular basis of recognition for a protein substrate is understood, extensive biochemical and genetic analysis have been performed, and single-molecule optical trapping has allowed direct visualization of the kinetics of substrate unfolding and translocation. In this review, we discuss our current understanding of ClpXP structure and function, evaluate competing sequential and probabilistic mechanisms of ATP hydrolysis, and highlight open questions for future exploration.

The structure of caseinolytic protease subunit ClpP2 reveals a functional model of the caseinolytic protease system from Chlamydia trachomatis.

Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved. Here, we report the first crystal structure of full-length ClpP2 as an inactive homotetradecamer in a complex with a candidate antibiotic at 2.66 Å resolution. The structure details the functional domains of the ClpP2 protein subunit and includes the handle domain, which is integral to proteolytic activation. In addition, hydrogen-deuterium exchange mass spectroscopy probed the dynamics of ClpP2, and molecular modeling of ClpP1 predicted an assembly with ClpP2. By leveraging previous enzymatic experiments, we constructed a model of ClpP2 activation and its interaction with the protease subunits ClpP1 and ClpX. The structural information presented will be relevant for future rational drug design against these targets and will lead to a better understanding of ClpP complex formation and activation within this important human pathogen.

Homozygous novel truncating variant of CLPP associated with severe Perrault syndrome.

A female proband and her affected niece are homozygous for a novel frameshift variant of CLPP. The proband was diagnosed with severe Perrault syndrome encompassing hearing loss, primary ovarian insufficiency, abnormal brain white matter and developmental delay.

New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila.

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, causes Legionnaires' disease in humans that is characterized by severe pneumonia. This bacterium adopts a distinct biphasic life cycle consisting of a nonvirulent replicative phase and a virulent transmissive phase in response to different environmental conditions. Hence, the timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial for survival and replication. Here, we report that the completion of the biphasic life cycle and bacterial pathogenesis is greatly dependent on the protein homeostasis regulated by caseinolytic protease P (ClpP)-dependent proteolysis. We characterized the ClpP-dependent dynamic profiles of the regulatory and substrate proteins during the biphasic life cycle of L. pneumophila using proteomic approaches and discovered that ClpP-dependent proteolysis specifically and conditionally degraded the substrate proteins, thereby directly playing a regulatory role or indirectly controlling cellular events via the regulatory proteins. We further observed that ClpP-dependent proteolysis is required to monitor the abundance of fatty acid biosynthesis-related protein Lpg0102/Lpg0361/Lpg0362 and SpoT for the normal regulation of L. pneumophila differentiation. We also found that the control of the biphasic life cycle and bacterial virulence is independent. Furthermore, the ClpP-dependent proteolysis of Dot/Icm (defect in organelle trafficking/intracellular multiplication) type IVB secretion system and effector proteins at a specific phase of the life cycle is essential for bacterial pathogenesis. Therefore, our findings provide novel insights on ClpP-dependent proteolysis, which spans a broad physiological spectrum involving key metabolic pathways that regulate the transition of the biphasic life cycle and bacterial virulence of L. pneumophila, facilitating adaptation to aquatic and intracellular niches.

Staphylococcal ClpXP protease targets the cellular antioxidant system to eliminate fitness-compromised cells in stationary phase.

The transition from growth to stationary phase is a natural response of bacteria to starvation and stress. When stress is alleviated and more favorable growth conditions return, bacteria resume proliferation without a significant loss in fitness. Although specific adaptations that enhance the persistence and survival of bacteria in stationary phase have been identified, mechanisms that help maintain the competitive fitness potential of nondividing bacterial populations have remained obscure. Here, we demonstrate that staphylococci that enter stationary phase following growth in media supplemented with excess glucose, undergo regulated cell death to maintain the competitive fitness potential of the population. Upon a decrease in extracellular pH, the acetate generated as a byproduct of glucose metabolism induces cytoplasmic acidification and extensive protein damage in nondividing cells. Although cell death ensues, it does not occur as a passive consequence of protein damage. Instead, we demonstrate that the expression and activity of the ClpXP protease is induced, resulting in the degeneration of cellular antioxidant capacity and, ultimately, cell death. Under these conditions, inactivation of either clpX or clpP resulted in the extended survival of unfit cells in stationary phase, but at the cost of maintaining population fitness. Finally, we show that cell death from antibiotics that interfere with bacterial protein synthesis can also be partly ascribed to the corresponding increase in clpP expression and activity. The functional conservation of ClpP in eukaryotes and bacteria suggests that ClpP-dependent cell death and fitness maintenance may be a widespread phenomenon in these domains of life.

Probing allosteric interactions in homo-oligomeric molecular machines using solution NMR spectroscopy.

Developments in solution NMR spectroscopy have significantly impacted the biological questions that can now be addressed by this methodology. By means of illustration, we present here a perspective focusing on studies of a number of molecular machines that are critical for cellular homeostasis. The role of NMR in elucidating the structural dynamics of these important molecules is emphasized, focusing specifically on intersubunit allosteric communication in homo-oligomers. In many biophysical studies of oligomers, allostery is inferred by showing that models specifically including intersubunit communication best fit the data of interest. Ideally, however, experimental studies focusing on one subunit of a multisubunit system would be performed as an important complement to the more traditional bulk measurements in which signals from all components are measured simultaneously. Using an approach whereby asymmetric molecules are prepared in concert with NMR experiments focusing on the structural dynamics of individual protomers, we present examples of how intersubunit allostery can be directly observed in high-molecular-weight protein systems. These examples highlight some of the unique roles of solution NMR spectroscopy in studies of complex biomolecules and emphasize the important synergy between NMR and other atomic resolution biophysical methods.

Mitochondrial Unfolded Protein Response Gene Clpp Is Required for Oocyte Function and Female Fertility.

Mitochondrial unfolded protein stress response (mtUPR) plays a critical role in regulating cellular and metabolic stress response and helps maintain protein homeostasis. Caseinolytic peptidase P (CLPP) is one of the key regulators of mtUPR and promotes unfolded protein degradation. Previous studies demonstrated that global deletion of Clpp resulted in female infertility, whereas no impairment was found in the mouse model with targeted deletion of Clpp in cumulus/granulosa cells. These results suggest the need to delineate the function of Clpp in oocytes. In this study, we aimed to further explore the role of mtUPR in female reproductive competence and senescence using a mouse model. Oocyte-specific targeted deletion of Clpp in mice resulted in female subfertility associated with metabolic and functional abnormalities in oocytes, thus highlighting the importance of CLPP-mediated protein homeostasis in oocyte competence and reproductive function.

A Photocrosslinking Probe to Capture the Substrates of Caseinolytic Protease P.

Protein homeostasis in bacteria is regulated by proteases such as the tetradecameric caseinolytic protease P (ClpP). Although substrates of ClpP have been successfully deciphered in genetically engineered cells, methods which directly trap processed proteins within native cells remain elusive. Here, we introduce an in situ trapping strategy which utilizes trifunctional probes that bind to the active site serine of ClpP and capture adjacent substrates with an attached photocrosslinking moiety. After enrichment using an alkyne handle, substrate deconvolution by mass spectrometry (MS) is performed. We show that our two traps bind substoichiometrically to ClpP, retain protease activity, exhibit unprecedented selectivity for Staphylococcus aureus ClpP in living cells and capture numerous known and novel substrates. The exemplary validation of trapped hits using a targeted proteomics approach confirmed the fidelity of this technology. In conclusion, we provide a novel chemical platform suited for the discovery of serine protease substrates beyond genetic engineering.

Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules.

ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a self-compartmentalizing protease composed of two stacked heptameric rings that allow protein degradation to occur within the catalytic chamber. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multilayered regulation of ClpP that may present novel ideas for structure-based drug design.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus.

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

Microbial Catabolic Activity: Methods, Pertinence, and Potential Interest for Improving Microbial Inoculant Efficiency.

Microbial catabolic activity (MCA) defined as the degrading activity of microorganisms toward various organic compounds for their growth and energy is commonly used to assess soil microbial function potential. For its measure, several methods are available including multi-substrate-induced respiration (MSIR) measurement which allow to estimate functional diversity using selected carbon substrates targeting specific biochemical pathways. In this review, the techniques used to measure soil MCA are described and compared with respect to their accuracy and practical use. Particularly the efficiency of MSIR-based approaches as soil microbial function indicators was discussed by (i) showing their sensitivity to different agricultural practices including tillage, amendments, and cropping systems and (ii) by investigating their relationship with soil enzyme activities and some soil chemical properties (pH, soil organic carbon, cation exchange capacity). We highlighted the potential of these MSIR-based MCA measurements to improve microbial inoculant composition and to determine their potential effects on soil microbial functions. Finally, we have proposed ideas for improving MCA measurement notably through the use of molecular tools and stable isotope probing which can be combined with classic MSIR methods. Graphical abstract describing the interrelation between the different parts and the concepts developed in the review.

CLPP deficiency ameliorates neurodegeneration caused by impaired mitochondrial protein synthesis.

Mitochondria are essential organelles found in every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in either mtDNA or nuclear DNA-encoded genes of mitochondrial proteins cause diseases affecting the oxidative phosphorylation system, which are heterogeneous from a clinical, genetic, biochemical and molecular perspective and can affect patients at any age. Despite all this, it is surprising that our understanding of the mechanisms governing mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored. We recently showed that loss of the mitochondrial matrix protease caseinolytic protease proteolytic subunit (CLPP) ameliorates phenotypes in cells characterized by defects in oxidative phosphorylation maintenance. Here, we build upon this finding by showing that CLPP depletion is indeed beneficial in vivo for various types of neuronal populations, including Purkinje cells in the cerebellum and cortical and hippocampal neurons in the forebrain, as it strongly improves distinct phenotypes of mitochondria encephalopathy, driven by the deficiency of the mitochondrial aspartyl tRNA synthase DARS2. In the absence of CLPP, neurodegeneration of DARS2-deficient neurons is delayed as they present milder oxidative phosphorylation dysfunction. This in turn leads to a decreased neuroinflammatory response and significantly improved motor functions in both double-deficient models (Purkinje cell-specific or forebrain neuron-specific Dars2/Clpp double knockout mice). We propose that diminished turnover of respiratory complex I caused by the loss of CLPP is behind the improved phenotype in Dars2/Clpp double knockout animals, even though this intervention might not restore respiratory complex I activity but rather improve mitochondrial cristae morphology or help maintain the NAD+/NADH ratio inside mitochondria. These results also open the possibility of targeting CLPP activity in many other mitochondrial encephalopathies characterized by respiratory complex I instability.

Unravelling the ultramafic rock-driven serpentine soil formation leading to the geo-accumulation of heavy metals: An impact on the resident microbiome, biogeochemical cycling and acclimatized eco-physiological profiles.

In the present study, we have underpinned the serpentine rock, serpentinized ultramafic soil and rhizosphere's microbial communities, signifying their heavy metals-exposed taxa signatures and functional repertoires in comparison to non-serpentine soils. The results revealed that the serpentine rock embedded soil highlighted the geo-accumulation of higher amount of Cr and Ni impacting soil microbial diversity negatively by metal stress-driven selection. Biolog Ecoplate CLPP defined a restricted spectrum of C-utilization in the higher heavy metal-containing serpentine samples compared to non-serpentine. The linear discriminant analysis (LDA) score identified a higher abundance of Desulfobacterota, Opitutales, and Bacteroidales in low Cr and Ni-stressed non-serpentine-exposed samples. Whereas the abundance of Propionibacteriales and Actinobacteriota were significantly enriched in the serpentine niche. Further, the C, N, S, Fe, and methane biogeochemical cycles linked functional members were identified, and showing higher functional diversity in low Cr and Ni concentration-containing rhizosphere JS-soils. The Pearson correlation coefficient (r) value confirmed the abundance of functional members linked to specific biogeochemical cycle, positively correlated with relevant pathway enrichment. Ultimately, this study highlighted the heavy metal stress within a serpentine setting that could limit the resident microbial community's metabolic diversity and further select the bacteria that could thrive in the serpentine-associated heavy metal-stressed soils. These acclimatized microbes could pave the way for the future applications in the soil conservation and management.

KPNB1 modulates the Machado-Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP.

Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD.

An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key ß-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.

Rapid sequence evolution is associated with genetic incompatibilities in the plastid Clp complex.

KEY MESSAGE: Replacing the native clpP1 gene in the Nicotiana plastid genome with homologs from different donor species showed that the extent of genetic incompatibilities depended on the rate of sequence evolution. The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.

ClpP1P2 peptidase activity promotes biofilm formation in Pseudomonas aeruginosa.

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP17 P27 tetradecamer that is required for proper biofilm development. PaClpP114 and PaClpP17 P27 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development.