Recombinant Chromobacterium violaceum ATP-dependent Clp protease proteolytic subunit (clpP)

What is the ATP-dependent Clp protease system in Chromobacterium violaceum?

The ATP-dependent Clp protease system in Chromobacterium violaceum is a multi-component proteolytic complex essential for protein quality control and regulation. Similar to Clp proteases in other bacteria, it consists of two main components: an ATP-dependent chaperone component (likely ClpC or ClpX) that recognizes, unfolds, and translocates substrate proteins, and a proteolytic core (containing ClpP) that degrades the unfolded proteins. The system uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites . This multisubunit chaperone-protease complex shares architectural similarities with other selective proteolysis systems and plays vital roles in cellular homeostasis.

How is the ClpP proteolytic subunit structurally organized?

The ClpP proteolytic subunit typically forms a barrel-shaped complex with multiple subunits arranged in two stacked heptameric rings, creating a sequestered chamber for proteolysis. Based on structural data from related systems, the C. violaceum ClpP likely follows this tetradecameric (14-subunit) architecture. Each ClpP subunit contains a catalytic triad (Ser-His-Asp) characteristic of serine proteases, which is essential for its proteolytic activity . The complex contains axial pores through which unfolded substrate proteins enter. These structural features are highly conserved across bacterial species, although some variations exist in the specific arrangement and composition of the proteolytic core.

What is the relationship between ClpP and pathogenicity in C. violaceum?

While the specific role of ClpP in Chromobacterium violaceum pathogenicity is not directly addressed in the provided search results, related research indicates that Clp proteases often play significant roles in bacterial virulence regulation. In C. violaceum, pathogenicity is primarily associated with the Type III Secretion System (T3SS) encoded by the Cpi-1 pathogenicity island, which is essential for the bacterium's interaction with host cells . The Clp protease system may be involved in regulating the expression or function of virulence factors, including those associated with the T3SS. The ClpP proteolytic subunit could potentially process key regulatory proteins involved in virulence gene expression, similar to its role in other bacterial pathogens.

What is the evolutionary significance of ClpP in the Chromobacterium genus?

The evolutionary perspective on ClpP within the Chromobacterium genus reveals its conservation and potential adaptation to different ecological niches. Chromobacterium species inhabit soil and freshwater environments but some, like C. violaceum, can cause opportunistic infections in humans and animals . The ClpP protein likely plays a role in adaptation to these diverse environments by regulating protein turnover and stress responses. Evolutionary analysis of ClpP sequences across the Chromobacterium genus could provide insights into adaptive changes associated with pathogenicity and environmental persistence. This evolutionary conservation underscores the fundamental importance of ClpP in bacterial physiology regardless of lifestyle.

What are effective strategies for recombinant expression of C. violaceum ClpP?

For recombinant expression of C. violaceum ClpP, researchers should consider the following evidence-based approaches:

• Vector Selection: Based on successful expression of related Clp proteins, pET-based expression vectors with T7 promoters are recommended for high-level expression in E. coli host systems .

• Co-expression Strategy: When working with multi-subunit complexes, co-expression of interacting partners often improves stability and solubility. For instance, if C. violaceum ClpP functions in a complex similar to the Synechococcus ClpP3/ClpR system, co-expression of partner proteins should be considered. The pACYC Duet vector system has proven effective for such co-expression approaches .

• Affinity Tag Placement: Addition of a C-terminal His6 tag has been demonstrated to be effective for purification while maintaining proper complex assembly, as shown in the successful purification of the Synechococcus ClpP3/R complex .

• Expression Conditions: Optimal expression typically occurs at lower temperatures (16-25°C) after induction, with extended expression times (12-18 hours) to promote proper folding and reduce inclusion body formation.

What purification methods yield the highest purity and activity of recombinant C. violaceum ClpP?

The purification of recombinant C. violaceum ClpP to achieve high purity and maintained activity requires a multi-step chromatographic approach:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged ClpP proteins. The recommended buffer composition includes Tris-HCl (pH 7.5-8.0), NaCl (200-300 mM), and imidazole gradient elution (20-250 mM) .

• Intermediate Purification: Size exclusion chromatography using Superdex 200 columns effectively separates correctly assembled ClpP complexes from aggregates and partially assembled intermediates, while simultaneously performing buffer exchange .

• Polishing Step: When necessary, ion exchange chromatography (particularly anion exchange using Q-Sepharose) can remove remaining contaminants based on charge differences.

• Activity Preservation: Throughout purification, inclusion of 10% glycerol, 1-5 mM DTT or β-mercaptoethanol, and performing all steps at 4°C significantly preserves proteolytic activity .

• Quality Assessment: Final preparation quality should be verified by SDS-PAGE (>95% purity), native PAGE (structural integrity), and peptidase activity assays using fluorogenic peptide substrates such as Suc-Leu-Tyr-AMC .

How can researchers troubleshoot expression and solubility issues with recombinant C. violaceum ClpP?

When encountering expression and solubility challenges with recombinant C. violaceum ClpP, implement this systematic troubleshooting framework:

• Expression Optimization:

  • Test multiple E. coli expression strains (BL21(DE3), Rosetta, Arctic Express)

  • Evaluate induction parameters (IPTG concentration: 0.1-1.0 mM; temperature: 16-37°C)

  • Implement auto-induction media which often improves yield for complex proteins

  • Consider codon optimization if expression levels remain low

• Solubility Enhancement:

  • Add solubility-enhancing tags (MBP, SUMO, TrxA) at the N-terminus

  • Include osmolytes (0.5-1.0 M sorbitol, 0.5-1.0 M trehalose) in lysis buffers

  • Test detergents (0.05-0.1% Triton X-100, 0.5-1.0% CHAPS) for improved extraction

  • Consider cell-free expression systems for particularly challenging cases

• Complex Formation Facilitation:

  • Co-express with potential protein partners (ClpX, ClpA, or ClpC chaperones)

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J) to improve folding

  • Test bicistronic constructs that mimic natural gene organization, similar to the successful ClpP3/R co-expression from Synechococcus

• Protein Stabilization:

  • Include stabilizing cofactors (5-10 mM MgCl₂) in all buffers

  • Test multiple pH conditions (pH 6.5-8.5) to identify optimal stability range

  • Add low concentrations of substrate analogs or inhibitors to stabilize active conformations

What methods are most effective for characterizing the oligomeric state of C. violaceum ClpP?

The oligomeric state of C. violaceum ClpP can be effectively characterized using multiple complementary techniques:

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): This technique provides accurate molecular weight determination of the native complex without shape assumptions, enabling precise determination of the oligomeric state. SEC-MALS has been successfully applied to related Clp complexes and allows detection of tetradecameric (~300 kDa) versus heptameric (~150 kDa) assemblies .

• Native Mass Spectrometry: This technique can resolve distinct oligomeric species and subunit stoichiometry with high precision. For heteromeric complexes like the ClpP3/R complex from Synechococcus, native MS was critical in determining the exact stoichiometry (ClpP3₃ClpR₄) of subunits within the heptameric rings .

• Analytical Ultracentrifugation (AUC): Sedimentation velocity and equilibrium experiments provide information about size, shape, and homogeneity of the complex in solution. AUC is particularly valuable for detecting concentration-dependent assembly/disassembly.

• Negative Stain Electron Microscopy: This visualization technique confirms the characteristic barrel-shaped architecture of assembled ClpP complexes and can detect structural heterogeneity in the sample.

• Chemical Crosslinking: In-solution crosslinking followed by SDS-PAGE analysis can capture and preserve oligomeric interactions, providing evidence of complex formation and subunit proximity.

How can researchers accurately assess the proteolytic activity of recombinant C. violaceum ClpP?

Accurate assessment of proteolytic activity of recombinant C. violaceum ClpP requires a multi-faceted approach that addresses both peptidase and protease activities:

• Peptidase Activity Assays:

  • Fluorogenic peptide substrates provide a sensitive and quantitative measure of peptidase activity

  • Recommended substrates include N-succinyl-Leu-Tyr-AMC, Suc-Val-Lys-Met-AMC, and Suc-Ile-Ile-Trp-AMC

  • Standard assay conditions: 30 μM peptide substrate, 1-5 μg ClpP, in buffer containing 25 mM Tris/Cl (pH 7.5), 75 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 37°C

  • Measure fluorescence using excitation/emission wavelengths of 310-380 nm/460 nm

• Protein Substrate Degradation:

  • Casein degradation assays using either unlabeled α-casein (monitored by SDS-PAGE) or FITC-casein (monitored by fluorescence)

  • GFP-tagged model substrates with specific degradation tags (FR-GFP, MR-GFP) to assess ATP-dependent degradation with partner chaperones

  • Standard assay conditions: 1 μM of each Clp protein component, 1 μM α-casein or 100 nM fluorescent substrates, with ATP regeneration system (5 mM ATP, 16 mM creatine phosphate, 10 μg/ml creatine kinase)

• Chaperone Partner Dependency:

  • Complete ClpP activity assessment requires testing with cognate chaperone partners (ClpX, ClpA, or ClpC) at 1:1 molar ratio

  • ATPase activity of the chaperone component should be measured in parallel to confirm functional coupling between ATP hydrolysis and proteolysis

• Comparative Activity Analysis:

  • Activity should be compared with well-characterized Clp proteases (e.g., E. coli ClpP) under identical conditions

  • Specific activity calculations (nmol substrate degraded per minute per mg enzyme) enable standardized comparison between preparations

What are the key structural differences between C. violaceum ClpP and ClpP from model organisms like E. coli?

The structural comparison between C. violaceum ClpP and the extensively studied E. coli ClpP reveals several important distinctions with functional implications:

While direct structural data for C. violaceum ClpP is not yet available, structural modeling based on sequence homology with E. coli ClpP suggests conservation of the core structural elements with potential variations in regions that interact with chaperone partners or substrates .

How can C. violaceum ClpP be exploited as a target for novel antimicrobial development?

The exploitation of C. violaceum ClpP as an antimicrobial target can be approached through several strategic pathways:

• ClpP Activation as Antimicrobial Strategy:

  • Acyldepsipeptide (ADEP) antibiotics represent a proven approach by dysregulating ClpP through conformational changes that open the axial gate and enable unregulated proteolysis

  • Structural analysis of C. violaceum ClpP binding to ADEP analogs could identify species-specific binding pocket variations for selective targeting

  • Combining ClpP activators with traditional antibiotics enhances efficacy against persister cells and biofilms

• ClpP Inhibition Approaches:

  • β-lactones have demonstrated efficacy as ClpP inhibitors by covalently modifying the active site serine

  • Development of transition-state analog inhibitors specific to C. violaceum ClpP's catalytic pocket

  • Peptide-based inhibitors that mimic natural degradation tags but resist proteolysis can compete for ClpP binding sites

• Chaperone-ClpP Interface Disruption:

  • Small molecules targeting the ClpX-ClpP or ClpC-ClpP interface disrupt crucial protein-protein interactions

  • This approach offers potential selectivity by targeting interfaces unique to C. violaceum

  • Structure-based design of peptidomimetics that interfere with the chaperone-protease docking

• Genetic Attenuation Applications:

  • ClpP deletion or controlled inhibition could be exploited for live-attenuated vaccine development against C. violaceum

  • Mouse infection models demonstrate that C. violaceum virulence mechanisms can be potentially regulated through proteolytic control systems

What role does ClpP play in the stress response and virulence regulation of C. violaceum?

Although direct experimental evidence specific to C. violaceum ClpP is limited in the provided search results, comprehensive analysis of related bacterial systems suggests several key roles for ClpP in stress response and virulence regulation:

• Stress Response Coordination:

  • ClpP likely serves as a central regulator for managing protein damage during environmental stress conditions

  • Heat shock, oxidative stress, and nutrient limitation typically trigger increased ClpP expression and activity

  • The proteolytic processing of stress-damaged proteins prevents toxic aggregation and recycles amino acids

• Virulence Factor Regulation:

  • In C. violaceum, the Cpi-1 T3SS is a major virulence determinant, and its components are likely subjected to ClpP-mediated quality control

  • Transcriptional regulators of virulence genes, including potential homologs of CilA (master regulator of Cpi-1/1a) and OhrR (important for C. violaceum virulence), may be regulated through controlled proteolysis

  • The protease-chaperone system likely maintains proper folding and assembly of secretion systems essential for pathogenicity

• Host-Pathogen Interaction Modulation:

  • The NLRC4 inflammasome recognizes C. violaceum T3SS components, triggering pyroptosis and bacterial clearance by neutrophils

  • ClpP-mediated proteolysis may regulate expression of bacterial factors that interact with host immune responses

  • Precise temporal regulation of virulence factor expression during infection progression often depends on proteolytic control systems

• Biofilm Formation and Antibiotic Tolerance:

  • ClpP likely influences biofilm development through degradation of regulatory proteins controlling extracellular matrix components

  • The protease contributes to antibiotic tolerance by regulating the persister cell formation pathway

  • In C. violaceum, these functions would be particularly relevant to its environmental persistence and opportunistic pathogenicity

What experimental approaches can identify the substrate specificity and physiological targets of C. violaceum ClpP?

Identifying the substrate specificity and physiological targets of C. violaceum ClpP requires integrating multiple experimental approaches:

• Proteome-wide Identification Strategies:

  • Trapped-substrate Approach: Expression of proteolytically inactive ClpP variants (S-A active site mutation) creates a substrate trap, allowing co-purification and identification of natural substrates

  • Quantitative Proteomics: Comparative proteomic analysis (TMT or SILAC) between wild-type and ΔclpP strains identifies proteins with altered abundance

  • Pulse-chase Degradomics: Metabolic labeling combined with time-course analysis reveals proteins with differential turnover rates dependent on ClpP

• Degradation Signal (Degron) Characterization:

  • Peptide Library Screening: Systematic analysis of fluorogenic peptide substrates to define sequence preferences

  • N-end Rule Analysis: Testing degradation of model substrates with different N-terminal residues reveals specificity patterns

  • C-terminal Tag Libraries: Systematic testing of SsrA-like tags and variants to define C. violaceum-specific recognition motifs

• Adaptor Protein Identification:

  • Bacterial Two-Hybrid Screening: Identification of proteins that interact with ClpP or its chaperone partners

  • Co-immunoprecipitation: Pulldown of ClpP complexes from C. violaceum lysates followed by mass spectrometry

  • Genomic Context Analysis: Identification of potential adaptor proteins (e.g., ClpS homologs) based on genomic proximity to clp genes

• In Vivo Validation Approaches:

  • Fluorescent Reporter Systems: GFP fusion libraries to monitor protein stability in vivo

  • Degron Transfer Experiments: Fusion of putative degradation signals to stable reporter proteins to confirm functionality

  • Conditional Degradation Systems: Engineering ClpP-dependent degradation tags for targeted protein depletion studies

How does the immune system recognize and respond to C. violaceum infection, and what role might ClpP play?

The complex interaction between the immune system and C. violaceum involves specific recognition mechanisms and defense strategies:

• Inflammasome-Mediated Recognition:

  • The NLRC4 inflammasome specifically recognizes the Cpi-1a T3SS needle protein CprI from C. violaceum

  • Human NAIP protein detects bacterial T3SS components and promotes NLRC4 inflammasome oligomerization

  • This recognition leads to caspase-1 activation and induces pyroptosis, a form of inflammatory cell death

  • ClpP may indirectly regulate this interaction by controlling the expression or assembly of T3SS components

• Multi-layered Defense Mechanisms:

  • In murine models, C. violaceum infection is controlled through two NLRC4-dependent pathways: pyroptosis and Natural Killer (NK) cell cytotoxicity

  • These mechanisms eject intracellular bacteria from macrophages and hepatocytes, exposing them to neutrophil killing

  • ClpP-regulated virulence factors likely influence the efficiency of bacterial escape from these defense mechanisms

• Neutrophil-Dependent Clearance:

  • Neutrophils play a critical role in the final elimination of C. violaceum

  • Patients with chronic granulomatous disease (CGD), who have neutrophil NADPH oxidase deficiencies, show dramatically increased susceptibility to C. violaceum infections

  • The bacterial ClpP system may regulate factors that provide resistance against neutrophil killing mechanisms

• Potential Therapeutic Implications:

  • Targeting ClpP could potentially enhance immune recognition or reduce bacterial evasion mechanisms

  • Modulating ClpP function might increase bacterial susceptibility to neutrophil-mediated killing

  • Understanding this host-pathogen interface provides opportunities for immunomodulatory therapeutic approaches

What regulatory networks govern C. violaceum virulence, and how might ClpP intersect with these pathways?

The regulatory networks governing C. violaceum virulence involve multiple interconnected pathways with potential ClpP intersection points:

• Transcriptional Regulation Hierarchies:

  • CilA functions as a master transcriptional activator of most Cpi-1/1a genes, which encode the T3SS essential for virulence

  • A cilA-mutant strain shows complete attenuation of virulence in mice, highlighting its crucial role

  • ClpP likely regulates the abundance or activity of transcription factors like CilA through controlled proteolysis

• Environmental Signal Integration:

  • OhrR, a MarR family transcriptional regulator, contributes significantly to C. violaceum virulence

  • Environmental signals that trigger virulence gene expression remain largely unknown

  • ClpP-mediated proteolysis often serves as a post-translational mechanism to integrate multiple environmental cues

• Quorum Sensing Connections:

  • Violacein production in C. violaceum is regulated by quorum sensing mechanisms

  • This system potentially coordinates population-level expression of virulence factors

  • ClpP may regulate quorum sensing by controlling the turnover of signal receptors or response regulators

• Post-transcriptional Control Systems:

  • Regulatory small RNAs and RNA-binding proteins often fine-tune virulence gene expression

  • ClpP could regulate these factors, adding another layer of control

  • Integrative analysis of transcriptomics and proteomics data from clpP mutants would reveal these regulatory intersections

What experimental systems can effectively model C. violaceum infection to study ClpP's role in pathogenesis?

Effective experimental systems for studying C. violaceum infection and ClpP's role in pathogenesis include:

• Mouse Models of Infection:

  • Systemic Infection Model: Intravenous injection of C. violaceum has been successfully used to study hepatic infection and systemic spread

  • Targeted Genetic Backgrounds: Wild-type mice effectively clear C. violaceum infection, while mice with specific immune defects (NLRC4-/-, Casp1/11-/-) show increased susceptibility

  • Chronic Granulomatous Disease Model: p47phox-/- mice mimic human CGD and provide a relevant model for studying neutrophil-bacteria interactions

• Cellular Infection Systems:

  • Macrophage Infection Assays: Primary bone marrow-derived macrophages or cell lines (RAW264.7, J774) for studying intracellular survival and inflammasome activation

  • Hepatocyte Models: Primary hepatocytes or hepatic cell lines for studying C. violaceum-induced cytotoxicity

  • Neutrophil Killing Assays: Isolated human or mouse neutrophils for evaluating bacterial susceptibility to neutrophil-mediated killing mechanisms

• Genetic Manipulation Approaches:

  • Inducible ClpP Depletion: Tetracycline-regulated expression systems allow controlled depletion of ClpP during different infection stages

  • Domain-specific Mutations: Strategic mutations in ClpP to disrupt specific functions while maintaining others

  • Complementation Analysis: Re-introduction of wild-type or mutant ClpP variants into ΔclpP strains to identify critical functional regions

• Ex Vivo Systems:

  • Precision-cut Liver Slices: Maintains tissue architecture while allowing controlled infection conditions

  • Microfluidic Organ-on-chip: Models incorporating immune cells and tissue cells provide dynamic infection environments

  • 3D Organoid Cultures: Liver or intestinal organoids offer physiologically relevant infection models with defined cell compositions

What emerging technologies could advance our understanding of C. violaceum ClpP structure and function?

Several cutting-edge technologies show particular promise for advancing our understanding of C. violaceum ClpP:

• Advanced Structural Biology Approaches:

  • Cryo-electron Microscopy: Near-atomic resolution structures of ClpP-chaperone complexes in different functional states

  • Time-resolved X-ray Crystallography: Capturing intermediate states during the proteolytic cycle

  • Hydrogen-Deuterium Exchange Mass Spectrometry: Mapping conformational changes and protein-protein interaction surfaces

  • Integrative Structural Biology: Combining multiple techniques (SAXS, NMR, EM) for complete structural characterization

• Single-Molecule Technologies:

  • Single-molecule FRET: Real-time observation of ClpP conformational dynamics during substrate processing

  • Optical Tweezers: Measuring forces during protein unfolding and translocation by the Clp complex

  • High-speed AFM: Visualizing structural changes in ClpP complexes during the proteolytic cycle

  • Nanopore Analysis: Direct observation of substrate translocation through the ClpP complex

• Systems Biology Approaches:

  • Genome-wide CRISPRi Screens: Identifying genetic interactions with clpP in C. violaceum

  • Multi-omics Integration: Combining transcriptomics, proteomics, and metabolomics to map ClpP's impact on cellular networks

  • Network Modeling: Predicting regulatory relationships and feedback loops involving ClpP

  • Machine Learning Applications: Predicting ClpP substrates based on integrated multi-omics datasets

• Advanced Genetic Tools:

  • Base Editing: Precise introduction of specific mutations in clpP without double-strand breaks

  • Proximity Labeling: BioID or APEX2 fusions to map the in vivo ClpP interactome

  • Degron Tagging: Auxin-inducible or other rapidly responsive degron systems for temporal control of ClpP levels

  • Cell-specific Promoters: Tissue-specific expression of C. violaceum genes in infection models

How could comparative studies of ClpP across Chromobacterium species inform evolutionary adaptations to different niches?

Comparative studies of ClpP across Chromobacterium species offer valuable insights into evolutionary adaptations:

• Phylogenetic Analysis Framework:

  • Reconstructing the evolutionary history of ClpP in the Chromobacterium genus reveals selective pressures

  • Comparing ClpP sequences from environmental versus clinical isolates identifies adaptations potentially related to pathogenicity

  • Analysis of selection signatures (dN/dS ratios) across different functional domains highlights regions under positive selection

  • Correlation between ClpP sequence variations and specific ecological niches or host ranges provides insights into adaptive specialization

• Functional Conservation and Divergence:

  • Heterologous expression of ClpP from different Chromobacterium species in standardized assay systems

  • Cross-complementation studies in ΔclpP mutant backgrounds to test functional interchangeability

  • Comparison of substrate specificities between ClpP variants from environmental and pathogenic isolates

  • Identification of species-specific adaptations in regulatory mechanisms controlling ClpP expression and activity

• Structural Implications of Sequence Variations:

  • Homology modeling of ClpP from multiple Chromobacterium species based on available crystal structures

  • Mapping sequence variations onto structural models to identify potentially functional differences

  • Comparative analysis of surface properties, particularly at interfaces with chaperone partners

  • Investigation of co-evolution patterns between ClpP and its chaperone partners across the genus

• Correlation with Pathogenicity Island Distribution:

  • The widespread occurrence of the Cpi-1 T3SS across Chromobacterium species suggests pathogenic potential throughout the genus

  • Investigating potential co-evolution between ClpP and virulence factors encoded by pathogenicity islands

  • Examining associations between specific ClpP variants and virulence in different host models

  • Tracing horizontal gene transfer events that may have influenced ClpP evolution in the genus