Recombinant Nitrosomonas europaea ATP-dependent Clp protease proteolytic subunit (clpP)

What is the ATP-dependent Clp protease proteolytic subunit (clpP) in Nitrosomonas europaea?

The ClpP in Nitrosomonas europaea is a serine protease that plays a crucial role in protein quality control and homeostasis. As part of the caseinolytic protease family, it functions by degrading misfolded, damaged, or regulatory proteins. Similar to ClpP in other bacteria, the N. europaea ClpP likely forms a tetradecameric complex consisting of two heptameric rings that create a barrel-shaped proteolytic chamber. This protease requires ATP-dependent chaperones (such as ClpX or ClpA) to recognize, unfold, and translocate substrate proteins into the proteolytic chamber where they are degraded into peptides of approximately 7-8 residues in length .

How does the ClpP protease system function in bacterial cells?

The ClpP protease system functions through a coordinated process involving both the ClpP proteolytic complex and AAA+ unfoldase partners. In bacteria like N. europaea, the process typically follows these steps:

• Substrate recognition: AAA+ unfoldases such as ClpX or ClpA recognize specific degradation tags or sequences on target proteins

• Complex formation: The unfoldase forms a hexameric complex that docks with the ClpP tetradecamer

• Protein unfolding: ATP hydrolysis powers the mechanical unfolding of substrate proteins

• Translocation: Unfolded proteins are threaded through the central pore into the proteolytic chamber

• Degradation: The serine protease activity of ClpP cleaves proteins into small peptide fragments

This mechanism allows for selective protein degradation, which is essential for various cellular processes including stress response, virulence regulation, and general proteostasis .

What is the significance of studying ClpP in Nitrosomonas europaea specifically?

Studying ClpP in Nitrosomonas europaea is particularly significant because:

• N. europaea is an environmentally important ammonia-oxidizing bacterium involved in the nitrogen cycle and nitrification processes

• As an obligate chemolithoautotroph, N. europaea has specialized metabolic systems that may reveal unique aspects of proteostasis regulation in these specialized bacteria

• N. europaea is important in wastewater treatment and has potential applications in bioremediation of sites contaminated with chlorinated hydrocarbons

• Understanding stress response mechanisms in N. europaea, in which ClpP likely plays a key role, can provide insights into how these bacteria adapt to environmental challenges

• ClpP-related genes in N. europaea (such as clpB) have been shown to upregulate in response to chlorinated compounds, suggesting their involvement in stress response pathways

Research on ClpP in this organism could therefore advance both fundamental understanding of bacterial proteostasis and applied fields such as environmental biotechnology and bioremediation.

What are the optimal conditions for expressing recombinant N. europaea ClpP in E. coli expression systems?

For optimal expression of recombinant N. europaea ClpP in E. coli expression systems, researchers should consider the following conditions:

Expression System Design:

• Vector selection: pET series vectors with T7 promoter systems typically provide high expression levels

• Host strain: BL21(DE3) or derivatives are recommended due to their deficiency in Lon and OmpT proteases

• Fusion tags: N-terminal His6-tag facilitates purification while minimally affecting protein structure

Expression Conditions:

• Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: 18-25°C post-induction (lower temperatures reduce inclusion body formation)

• Duration: 16-20 hours at lower temperatures or 3-4 hours at 37°C

• Media: Enriched media (such as Terrific Broth) supplemented with glucose (0.5-1%)

This approach is based on successful expression strategies for proteases from similar bacterial species, though specific optimization may be necessary for N. europaea ClpP. The relatively low post-induction temperatures help maintain protein solubility and proper folding, which is particularly important for proteases that might otherwise form inactive aggregates .

What purification strategy yields the highest activity for recombinant N. europaea ClpP?

A multi-step purification strategy is recommended to obtain highly active recombinant N. europaea ClpP:

Step 1: Initial Capture

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Wash with increasing imidazole (20-50 mM) to remove non-specifically bound proteins

• Elute with 250-300 mM imidazole gradient

Step 2: Intermediate Purification

• Ion exchange chromatography (IEX) using Q-Sepharose

• Buffer: 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol

• Elute with 50-500 mM NaCl gradient

Step 3: Polishing

• Size exclusion chromatography using Superdex 200

• Buffer: 25 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol, 1 mM DTT

Critical Considerations:

• Maintain temperature at 4°C throughout purification

• Include protease inhibitors (except serine protease inhibitors) in early purification steps

• Verify oligomeric state (tetradecamer formation) through size exclusion chromatography

• Assess activity using fluorogenic peptide substrates (such as Suc-LY-AMC)

This strategy typically yields protein with >95% purity and specific activity comparable to native enzyme. The tetradecameric assembly of ClpP is essential for its activity, so conditions that promote proper oligomerization are crucial for maintaining enzyme function .

How can researchers verify the correct oligomeric assembly of recombinant N. europaea ClpP?

Verification of the correct oligomeric assembly of recombinant N. europaea ClpP is critical since the tetradecameric structure is essential for proper function. Multiple complementary techniques should be employed:

Analytical Size Exclusion Chromatography (SEC):

• Use calibrated Superdex 200 or similar column

• Expected molecular weight: ~300 kDa for tetradecamer

• Buffer conditions: 25 mM HEPES pH 7.5, 150 mM KCl

• Compare elution profile with known standards

Native PAGE Analysis:

• 4-12% gradient gels recommended

• Compare migration pattern with other characterized ClpP proteins

Dynamic Light Scattering (DLS):

• Measures hydrodynamic radius

• Expected diameter: ~10-12 nm for tetradecamer

• Can detect presence of aggregates or monomeric species

Transmission Electron Microscopy (TEM):

• Negative staining with uranyl acetate

• Expected morphology: hollow barrel-shaped structures with dimensions ~9 nm × 9 nm

Analytical Ultracentrifugation (AUC):

• Sedimentation velocity experiments

• Expected sedimentation coefficient: ~11-12S for tetradecamer

Table 1: Expected Parameters for N. europaea ClpP Oligomeric States

The tetradecameric assembly can be sensitive to salt concentration, pH, and protein concentration, so these parameters should be systematically tested when optimizing conditions for structural studies and activity assays .

What are the most reliable methods for measuring N. europaea ClpP proteolytic activity in vitro?

Several complementary methods can be used to reliably measure the proteolytic activity of recombinant N. europaea ClpP in vitro:

Fluorogenic Peptide Substrates:

• Primary assay: Use of small fluorogenic peptide substrates such as Suc-LY-AMC

• Buffer: 50 mM Tris-HCl pH 8.0, 100 mM KCl, 5 mM MgCl2, 1 mM DTT

• Excitation/emission: 380/460 nm

• Concentration range: 0.1-1 mM substrate

• Controls: Include PMSF (1 mM) as negative control to verify serine protease activity

Coupled Assay with AAA+ Unfoldases:

• For full physiological activity assessment, include purified ClpX or ClpA

• Substrate: GFP-ssrA or FITC-casein (5-10 μM)

• ATP requirement: 4 mM ATP, 10 mM MgCl2, ATP regeneration system

• Measure fluorescence decrease over time as structured proteins are degraded

Gel-Based Assays:

• SDS-PAGE analysis of degradation of model substrates

• Time-course sampling (0-60 minutes)

• Visualization by Coomassie or silver staining

• Quantification by densitometry

Enzymatic Parameters:

• Determine Km and kcat using varying substrate concentrations

• Establish optimal temperature (likely 30-37°C) and pH (likely 7.5-8.5)

• Assess effects of various divalent cations (Mg2+, Mn2+, Ca2+)

The combination of these approaches provides a comprehensive profile of ClpP activity, with the fluorogenic peptide assay offering quick quantitative measures and the coupled assays providing insights into the physiologically relevant protein degradation capacity when partnered with appropriate unfoldases .

How does N. europaea ClpP specificity compare to ClpP from other bacterial species?

Comparing N. europaea ClpP specificity to ClpP from other bacterial species reveals both conserved features and potential differences:

Substrate Size and Structure:
Like other bacterial ClpP proteins, N. europaea ClpP likely demonstrates:

• Primary specificity for hydrophobic residues at the P1 position (particularly Leu, Phe, and Tyr)

• Peptidase activity for small peptides (7-10 residues) without requiring unfoldases

• Dependence on AAA+ unfoldases for degradation of larger proteins

Comparing Cleavage Preferences:
While specific data for N. europaea ClpP is limited, comparative analysis with model bacterial ClpP suggests:

Potential Unique Features:
As an ammonia-oxidizing bacterium with specialized metabolism, N. europaea ClpP may have evolved specific features:

• Potential adaptations for stress response to environmental contaminants

• Possible role in regulating proteins involved in ammonia oxidation pathway

• Specificity that may reflect the unusual proteome composition of this chemolithoautotroph

Studies using peptide libraries and proteomic approaches would be needed to definitively map the cleavage specificity of N. europaea ClpP and identify its natural substrates. Understanding these specificities could provide insights into the specialized role of protein quality control in ammonia-oxidizing bacteria .

What are the key factors affecting the stability and activity of recombinant N. europaea ClpP?

Several key factors significantly affect the stability and activity of recombinant N. europaea ClpP:

Buffer Composition:

• pH: Optimal range likely 7.5-8.0; activity decreases sharply below pH 6.5 or above pH 9.0

• Ionic strength: 100-200 mM monovalent salts (KCl or NaCl) typically optimal

• Divalent cations: Mg2+ (1-5 mM) essential for optimal activity

• Reducing agents: 1-5 mM DTT or 2-10 mM β-mercaptoethanol helps maintain cysteine residues

Temperature Effects:

• Storage stability: Significant activity loss after prolonged storage above 4°C

• Thermal stability: Typical denaturation temperatures range from 45-55°C

• Activity temperature: Optimal activity likely corresponds to N. europaea growth temperature (25-30°C)

Protein Concentration:

• Dilution effects: Activity decreases at concentrations below 50-100 nM due to dissociation of tetradecamer

• Concentration for storage: 0.5-1 mg/ml in storage buffer with 10-20% glycerol

Chemical Stability:

• Oxidation sensitivity: Surface-exposed cysteines may affect long-term stability

• Protease susceptibility: Self-cleavage can occur during prolonged storage

Additives for Stability:

• Glycerol (10-20%): Enhances long-term stability

• BSA (0.1 mg/ml): Prevents surface adsorption

• EDTA (0.5-1 mM): Inhibits metal-dependent proteases that may contaminate preparations

Long-term Storage Recommendations:

• Flash-freeze aliquots in liquid nitrogen

• Store at -80°C for long-term stability

• Avoid repeated freeze-thaw cycles (limit to ≤3)

Understanding these factors is essential for maintaining consistent enzyme activity in biochemical and structural studies. The tetradecameric structure of ClpP is particularly sensitive to buffer conditions, which can lead to disassembly into heptameric rings or monomers with consequent loss of activity .

How can researchers engineer N. europaea ClpP for enhanced activity or altered specificity?

Engineering N. europaea ClpP for enhanced activity or altered specificity can be approached through several rational design and directed evolution strategies:

Site-Directed Mutagenesis Approaches:

• Active site engineering: Mutations in the catalytic triad (Ser-His-Asp) can alter reactivity and substrate preference

• Substrate binding pocket modifications: Changes to hydrophobic residues lining the active site can alter P1 specificity

• Axial channel engineering: Modifications to residues lining the entry pores can alter substrate size restrictions

• Interface engineering: Mutations at subunit interfaces can stabilize the tetradecameric assembly

Structural Regions for Targeted Engineering:

• Catalytic triad: Typically Ser-His-Asp (positions determined by structural alignment)

• Hydrophobic pocket: Residues surrounding the S1 specificity pocket

• Active site adjacent loops: Can be modified to alter substrate positioning

• Oligomerization interfaces: Residues at heptamer-heptamer and subunit-subunit contacts

Directed Evolution Strategies:

• Error-prone PCR to generate libraries with random mutations

• DNA shuffling with ClpP from related species to generate chimeric enzymes

• Selection using fluorogenic substrates or growth-based selection systems

• Phage display methods to select variants with desired binding properties

Potential Applications of Engineered Variants:

• Biosensors: Engineering ClpP to respond to specific environmental contaminants relevant to N. europaea ecology

• Enhanced thermostability: For improved industrial applications

• Altered specificity: To target specific problem proteins or peptides

• Controlled activation: Engineering variants responsive to specific activators

Table 2: Potential Engineering Targets in ClpP Based on Known Structural Features

*Positions estimated based on homology with E. coli ClpP

Engineering approaches should consider maintaining the critical oligomeric structure of ClpP, as alterations that disrupt tetradecamer formation will likely abolish activity .

What is the role of ClpP in stress response of N. europaea to environmental contaminants?

The role of ClpP in stress response of Nitrosomonas europaea to environmental contaminants appears to be significant, though research in this specific area is still developing:

Evidence from Related Clp Proteins:
Studies with N. europaea have shown that the related ClpB protein is significantly upregulated in response to chloroform exposure. In transformed N. europaea with a GFP reporter driven by the clpB promoter, researchers observed a 6-10 fold increase in fluorescence when exposed to chloroform concentrations of 28-100 μM . This suggests that the Clp protease system, which includes ClpP, plays an important role in the cellular response to environmental stressors.

Potential Mechanisms in Contaminant Response:

• Degradation of damaged proteins: Chlorinated compounds can cause protein damage through oxidative stress

• Remodeling of the proteome: Targeted degradation of specific proteins to adapt metabolism

• Regulation of stress response pathways: Control of transcription factors involved in stress response

• Clearance of protein aggregates: Removal of misfolded proteins resulting from chemical stress

Specific Environmental Contaminants:
N. europaea encounters various environmental contaminants due to its ecological niche:

• Chlorinated aliphatic compounds (as studied with ClpB response)

• Heavy metals present in wastewater

• Aromatic compounds in industrial waste

• Agricultural chemicals including pesticides

Implications for Bioremediation:
Understanding ClpP's role in stress response has implications for bioremediation applications:

• N. europaea has potential for bioremediation of sites contaminated with chlorinated hydrocarbons

• The stress response mediated by the Clp system may be crucial for maintaining cell viability during bioremediation

• Engineering ClpP could potentially enhance N. europaea's tolerance to specific contaminants

Future research should explore the specific substrates of ClpP under different stress conditions and determine whether manipulation of ClpP activity could enhance N. europaea's resilience to environmental contaminants, potentially improving its utility in bioremediation applications .

How does ClpP function affect the ammonia oxidation pathway in N. europaea?

The relationship between ClpP function and the ammonia oxidation pathway in N. europaea represents an important but understudied area of research. Based on what is known about ClpP function in other bacteria and the unique metabolism of N. europaea, several potential interactions can be proposed:

Regulation of Key Metabolic Enzymes:
As a proteolytic enzyme involved in protein quality control, ClpP likely affects the turnover of key enzymes in the ammonia oxidation pathway:

• Ammonia monooxygenase (AMO): The central enzyme in ammonia oxidation may be subject to ClpP-mediated quality control

• Hydroxylamine oxidoreductase (HAO): Another crucial enzyme that may be regulated by proteolysis

• Electron transport proteins: Components of the electron transport chain that may be degraded when damaged by oxidative stress

Response to Metabolic Stress:
N. europaea's obligate chemolithoautotrophic lifestyle presents unique challenges:

• Energy limitation: When ammonia is scarce, ClpP may help reallocate cellular resources

• Oxidative stress: Ammonia oxidation generates reactive oxygen species that can damage proteins

• Carbon limitation: When CO2 fixation is challenged, ClpP may help manage protein economy

Potential Integration with Regulatory Networks:

• Transcription factors: ClpP may degrade transcriptional regulators that control expression of ammonia oxidation genes

• Signaling proteins: Sensors and signal transduction components could be ClpP substrates

• Stress response integration: Coordination between ammonia oxidation rate and stress response systems

Hypothesized Model of ClpP Influence:
During normal growth, ClpP likely maintains proteostasis by removing damaged proteins. During stress conditions (environmental contaminants, ammonia limitation, etc.), ClpP activity might increase to:

• Remove damaged enzymes from the ammonia oxidation pathway

• Adjust the proteome to conserve energy

• Recalibrate metabolic flux by altering enzyme levels

Research Gaps and Future Directions:

• Proteomic analysis of ClpP substrates under different growth conditions

• Construction of conditional ClpP mutants to assess effects on ammonia oxidation

• Investigation of how ClpP activity responds to changes in ammonia concentration

• Study of potential regulatory interactions between ClpP and transcription factors controlling ammonia oxidation genes

Understanding these interactions could provide insights into how this environmentally important bacterium balances its central metabolism with stress response and adaptation to changing environmental conditions .

What is the recommended protocol for cloning the ClpP gene from N. europaea?

The following protocol is recommended for cloning the ClpP gene from Nitrosomonas europaea, considering the organism's specific characteristics and the requirements for subsequent expression studies:

Materials Required:

• N. europaea ATCC 19718 culture

• High-fidelity DNA polymerase (Q5 or Phusion)

• Restriction enzymes compatible with expression vector

• T4 DNA ligase

• E. coli cloning strain (DH5α or TOP10)

• Expression vector (pET-28a or similar)

Protocol Steps:

• Genomic DNA Extraction

  • Culture N. europaea in minimal medium with 25 mM ammonium sulfate at 28°C

  • Harvest cells at late log phase (OD600 ~0.6-0.8)

  • Extract genomic DNA using a commercial kit optimized for Gram-negative bacteria

  • Verify DNA quality by agarose gel electrophoresis (0.8% gel)

• PCR Amplification

  • Design primers based on the N. europaea genome sequence (GenBank accession number: NC_004757)

  • Forward primer should include NdeI site and start codon

  • Reverse primer should include BamHI site and exclude stop codon for C-terminal tagging

  • PCR conditions:

    • Initial denaturation: 98°C for 30 seconds

    • 30 cycles: 98°C for 10 seconds, 60-65°C for 30 seconds, 72°C for 45 seconds

    • Final extension: 72°C for 2 minutes

• Restriction Digestion and Ligation

  • Digest PCR product and vector with NdeI and BamHI

  • Gel purify digested products (1% agarose)

  • Ligate using T4 DNA ligase (16°C overnight)

  • Transform into E. coli DH5α using heat shock method

• Clone Verification

  • Screen colonies by colony PCR

  • Verify positive clones by restriction digestion

  • Confirm sequence by Sanger sequencing

  • Check for complete ORF and absence of mutations

• Subcloning for Expression

  • Transfer verified construct to expression vector (pET-28a recommended)

  • Transform into expression strain (BL21(DE3) or Rosetta)

  • Verify construct by restriction analysis and sequencing

Critical Considerations:

• N. europaea has a high GC content genome (~50.7%), which may require DMSO (5%) in PCR reactions

• Codon optimization might be necessary if rare codons are present in the N. europaea sequence

• Consider cloning both N-terminal and C-terminal His-tagged versions for flexibility in purification approaches

• For structural studies, include a precision protease cleavage site between the tag and ClpP sequence

This protocol can be adapted based on specific experimental requirements and the intended applications of the recombinant protein .

What are the best approaches for studying ClpP-substrate interactions in N. europaea?

Multiple complementary approaches can be employed to study ClpP-substrate interactions in Nitrosomonas europaea:

In Vitro Biochemical Approaches:

• Direct Binding Studies

  • Surface plasmon resonance (SPR) with immobilized ClpP or substrate

  • Isothermal titration calorimetry (ITC) to determine binding thermodynamics

  • Microscale thermophoresis (MST) for solution-based interaction measurements

  • Fluorescence anisotropy with labeled peptide substrates

• Activity-Based Substrate Identification

  • Design of activity-based probes that covalently modify the active site

  • Fluorogenic substrates with systematic amino acid substitutions to map specificity

  • In vitro degradation assays with purified candidate substrates

  • Reconstituted systems with ClpP and cognate AAA+ unfoldases

Proteomic Approaches:

• Global Substrate Identification

  • SILAC or TMT-based quantitative proteomics comparing wild-type and ClpP-depleted strains

  • Pulse-chase experiments with stable isotope labeling to measure protein turnover rates

  • Protein trapping approaches using inactive ClpP (S97A mutant) as substrate trap

  • Proximity labeling methods using ClpP fused to promiscuous biotin ligase

• Crosslinking Mass Spectrometry

  • In vivo crosslinking followed by ClpP immunoprecipitation

  • MS/MS analysis to identify crosslinked peptides

  • Structural mapping of interaction sites

Genetic and Cellular Approaches:

• Conditional Depletion Studies

  • Construction of conditional ClpP mutants using inducible promoters

  • Quantitative proteomics following ClpP depletion

  • Phenotypic analysis under different stress conditions

• Fluorescence-Based Cellular Assays

  • GFP-fusion libraries to monitor protein stability

  • Split fluorescent protein complementation to detect interactions

  • FRET-based sensors for monitoring ClpP activity in vivo

Table 3: Comparative Analysis of ClpP-Substrate Interaction Methods

When interpreting results, it's important to differentiate between direct ClpP substrates and indirect effects caused by altered proteostasis. Validation across multiple methods provides the strongest evidence for bona fide substrate identification .

How can researchers develop assays to measure N. europaea ClpP activity in vivo?

Developing assays to measure N. europaea ClpP activity in vivo presents unique challenges due to the specialized metabolism and growth characteristics of this ammonia-oxidizing bacterium. The following approaches can be implemented:

Fluorescent Reporter Systems:

• Degradation Tag-Based Reporters

  • Construction: Fuse known ClpP degradation tags (ssrA, N-end rule) to fluorescent proteins (GFP, mCherry)

  • Implementation: Transform N. europaea with these constructs using established methods

  • Measurement: Monitor fluorescence intensity using flow cytometry or fluorescence microscopy

  • Analysis: Higher fluorescence indicates lower ClpP activity (less degradation of reporter)

• Split Fluorescent Protein Systems

  • Design: Fuse one fragment to ClpP and another to a known substrate

  • Principle: Fluorescence occurs during interaction before degradation

  • Advantage: Provides spatial information about ClpP activity in cells

Proteolytic Activity Probes:

• Activity-Based Probes

  • Develop cell-permeable fluorescent probes that covalently bind to active ClpP

  • These probes typically contain:

    • A reactive group that binds the active site serine

    • A fluorophore for detection

    • A specificity element that targets ClpP over other proteases

  • Cellular extracts can then be analyzed by SDS-PAGE with fluorescence imaging

• FRET-Based Sensors

  • Design: Construct sensors with ClpP recognition sequence between FRET pairs

  • Measurement: Changes in FRET ratio indicate cleavage by ClpP

  • Advantage: Potential for real-time measurements in living cells

Genetic Approaches:

• Conditional Mutant Analysis

  • Generate temperature-sensitive or chemically regulated ClpP variants

  • Monitor proteome changes using quantitative proteomics

  • Track phenotypic changes under stress conditions relevant to N. europaea ecology

• Substrate Stabilization Assay

  • Identify putative ClpP substrates with degradation tags

  • Monitor their abundance in wild-type vs. ClpP-depleted cells

  • Validate using Western blotting or targeted proteomics

Implementation Considerations for N. europaea:

• Transformation Efficiency

  • Use electroporation methods optimized for N. europaea

  • Consider integrating constructs into chromosome for stability

• Growth Rate Challenges

  • N. europaea grows slowly (doubling time ~8-12 hours)

  • Design experiments with appropriate time scales

  • Include proper controls for growth phase effects

• Promoter Selection

  • Use well-characterized N. europaea promoters for consistent expression

  • Consider inducible systems when available

  • The amoC promoter or the promoter used in the ClpB study may be suitable

• Environmental Factors

  • Test activity under relevant environmental stressors (chlorinated compounds, ammonia limitation)

  • Consider the effects of oxidative stress from ammonia oxidation

These approaches can be combined to provide complementary data on ClpP activity under different conditions, offering insights into the role of this protease in N. europaea physiology and stress response .

How does N. europaea ClpP compare structurally and functionally with ClpP from other bacterial species?

N. europaea ClpP exhibits both conservation and unique features when compared with ClpP from other bacterial species:

Structural Comparison:
Based on homology modeling and known bacterial ClpP structures, N. europaea ClpP likely shares these key structural elements:

• Core Fold and Oligomeric Structure

  • Tetradecameric assembly (two heptameric rings)

  • α/β fold with central serine protease active site

  • Handle region mediating ring-ring interactions

• Active Site Architecture

  • Canonical Ser-His-Asp catalytic triad

  • Oxyanion hole for stabilizing transition state

  • Hydrophobic S1 pocket for substrate binding

Table 4: Structural Comparison of ClpP Across Bacterial Species

*Based on sequence homology and predicted features

Functional Comparison:

• Substrate Specificity

  • Primary specificity for hydrophobic residues at P1 position (shared with other ClpPs)

  • Potential specialization for substrates relevant to ammonia oxidation metabolism

  • Predicted peptide products of 7-8 residues in length, similar to other bacterial ClpPs

• Unfoldase Interactions

  • As an obligate chemolithoautotroph, N. europaea has a more specialized proteome

  • Unfoldase partners may include specialized adaptations for regulating stress responses

• Regulation

  • Likely regulated by nutrient availability and stress conditions

  • May have evolved specific regulatory mechanisms to function in the context of ammonia oxidation

  • Might respond to environmental stressors like chlorinated compounds, similar to ClpB

Evolutionary Context:
N. europaea belongs to the betaproteobacteria, and its ClpP likely reflects evolutionary adaptations to its specialized niche:

• Conservation of core mechanisms of protein quality control

• Potential specialization for the unique cellular environment of an ammonia oxidizer

• Adaptations for stress conditions encountered in its ecological niche

Understanding these similarities and differences can inform research on the specific roles of ClpP in N. europaea metabolism and stress response, particularly in relation to its ammonia-oxidizing lifestyle and environmental adaptation mechanisms .

What is known about the regulation of ClpP expression in N. europaea under different environmental conditions?

The regulation of ClpP expression in Nitrosomonas europaea under different environmental conditions is not extensively documented, but insights can be drawn from related studies on stress response in this organism and general patterns of ClpP regulation in bacteria:

Evidence from Related Clp Proteins:
Studies on ClpB in N. europaea have shown significant upregulation in response to specific stressors:

• Chloroform exposure: ClpB promoter activity increased 6-10 fold in response to 28-100 μM chloroform

• No response to hydrogen peroxide: Unlike other Clp proteins, ClpB did not respond to oxidative stress from H2O2

This differential response suggests specialized regulation of Clp proteins in N. europaea that may extend to ClpP as well.

Predicted Regulatory Mechanisms:

• Transcriptional Regulation

  • Stress-responsive sigma factors likely control clpP expression

  • Potential binding sites for transcriptional regulators in the clpP promoter region

  • Integration with global stress response networks

• Environmental Triggers for ClpP Expression

  • Ammonia limitation: As an obligate ammonia oxidizer, N. europaea likely upregulates protein quality control under nutrient limitation

  • Temperature stress: ClpP is typically heat-shock regulated in bacteria

  • Chemical stressors: Chlorinated compounds and other environmental contaminants may induce expression

• Metabolic Integration

  • Carbon limitation: As an autotroph, N. europaea faces unique challenges in carbon acquisition

  • Energy stress: Limited energy from ammonia oxidation may trigger proteolytic responses

  • Oxidative stress: Ammonia oxidation generates reactive oxygen species that may damage proteins

Table 5: Predicted Regulatory Responses of N. europaea ClpP Under Different Conditions

Research Gaps and Future Directions:

• Transcriptomic analysis of N. europaea under various stressors to determine clpP expression patterns

• Reporter gene constructs similar to those used for clpB to monitor clpP promoter activity

• Investigation of potential unique regulatory mechanisms related to ammonia oxidation

• Identification of transcription factors that regulate clpP expression

This knowledge would contribute to understanding how this environmentally important bacterium maintains proteostasis under changing conditions and potential stress factors encountered in its ecological niche or during bioremediation applications .

How can evolutionary analysis of ClpP sequences inform functional studies in N. europaea?

Evolutionary analysis of ClpP sequences can provide valuable insights for functional studies in Nitrosomonas europaea by revealing conserved features, lineage-specific adaptations, and potential functional specializations:

Sequence Conservation Analysis:

• Identifying Functionally Critical Residues

  • Catalytic triad residues (Ser-His-Asp) will show absolute conservation

  • Substrate-binding pocket residues may show varying degrees of conservation

  • Residues involved in oligomerization interfaces are typically highly conserved

• Detection of Lineage-Specific Adaptations

  • Residues under positive selection may indicate adaptation to specific environmental niches

  • N. europaea-specific or ammonia oxidizer-specific substitutions may reveal functional specialization

  • Comparison with other chemolithoautotrophs vs. heterotrophs can highlight metabolic adaptations

Phylogenetic Approaches:

• Constructing ClpP Phylogenetic Trees

  • Position of N. europaea ClpP relative to other bacterial ClpPs

  • Evolutionary distance to other proteobacterial ClpPs

  • Clustering patterns that may indicate functional convergence

• Rate of Evolution Analysis

  • Accelerated evolution in specific lineages may indicate functional divergence

  • Slower evolution suggests strong selective constraints on function

Structure-Function Predictions:

• Homology Modeling Informed by Evolution

  • Identify suitable structural templates based on evolutionary relationships

  • Map conserved vs. variable regions onto structural models

  • Predict impact of N. europaea-specific residues on function

• Coevolution Analysis

  • Identify residues that show correlated evolutionary patterns

  • Predict functional interactions and structural contacts

  • Detect coevolutionary patterns with potential AAA+ unfoldase partners

Table 6: Evolutionary Analysis Approaches and Their Applications to N. europaea ClpP

Practical Implementation for N. europaea ClpP Studies:

• Database Construction

  • Gather ClpP sequences from diverse bacterial phyla

  • Include multiple sequences from ammonia-oxidizing bacteria

  • Incorporate sequences from bacteria with similar ecological niches

• Analytical Pipeline

  • Multiple sequence alignment using MAFFT or similar tools

  • Phylogenetic tree construction using maximum likelihood methods

  • Positive selection analysis using PAML or similar packages

  • Structural mapping of results using PyMOL or similar visualization tools

• Interpretation Framework

  • Map evolutionary findings to functional hypotheses

  • Prioritize residues for site-directed mutagenesis

  • Design chimeric proteins based on evolutionary insights

This evolutionary perspective can guide experimental design, help interpret functional data, and reveal adaptations that may be relevant to N. europaea's unique ecological niche as an ammonia-oxidizing bacterium .

What are the most promising future research directions for studying recombinant N. europaea ClpP?

The study of recombinant Nitrosomonas europaea ClpP presents several promising research directions that could advance both fundamental understanding of protein quality control in chemolithoautotrophs and applied aspects of environmental biotechnology:

Fundamental Mechanistic Studies:

• Structure-Function Relationships

  • Determination of high-resolution structure of N. europaea ClpP

  • Comparison with ClpP structures from heterotrophic bacteria

  • Investigation of potential structural adaptations related to ammonia oxidation lifestyle

• Proteome-Wide Substrate Identification

  • Global identification of ClpP substrates in N. europaea

  • Correlation with proteins involved in ammonia oxidation and carbon fixation

  • Mapping degradation signals (degrons) specific to N. europaea ClpP

• Regulatory Network Integration

  • Elucidation of transcriptional and post-translational regulation of ClpP

  • Mapping interactions between ClpP and stress response pathways

  • Understanding coordination between proteostasis and core metabolism

Applied Research Directions:

• Environmental Sensing Applications

  • Development of ClpP-based biosensors for environmental contaminants

  • Extension of the ClpB biosensor approach to monitor various stressors

  • Integration with field-deployable detection systems

• Bioremediation Enhancement

  • Engineering ClpP to improve N. europaea tolerance to toxic compounds

  • Developing strains with enhanced proteostasis for bioremediation applications

  • Exploring potential for improved ammonia removal in wastewater treatment

• Synthetic Biology Applications

  • Using ClpP as a controllable proteolytic module in synthetic circuits

  • Engineering degradation tags for temporal control of protein function

  • Development of tunable protein quality control systems

Technological Innovations:

• Advanced Imaging Approaches

  • Single-molecule tracking of ClpP function in living N. europaea cells

  • Super-resolution microscopy to localize ClpP relative to ammonia oxidation machinery

  • Time-lapse studies of ClpP dynamics during stress response

• High-Throughput Screening Platforms

  • Development of assays for ClpP modulators

  • Screening for compounds that enhance bioremediation capacity

  • Discovery of specific inhibitors or activators

• Integrative Multi-Omics Approaches

  • Combining proteomics, transcriptomics, and metabolomics

  • Systems biology modeling of ClpP's role in cellular homeostasis

  • Machine learning applications to predict ClpP function and regulation

These research directions would not only enhance our understanding of this specific protease but could also provide broader insights into protein quality control in environmentally important bacteria and potentially lead to biotechnological applications in environmental monitoring and remediation .

What are the key challenges in working with recombinant N. europaea ClpP and how can they be overcome?

Working with recombinant Nitrosomonas europaea ClpP presents several significant challenges, from expression and purification to functional analysis. Here, we identify these challenges and propose strategies to overcome them:

Expression and Purification Challenges:

• Low Expression Yields

  • Challenge: N. europaea proteins often express poorly in heterologous systems

  • Solutions:

    • Codon optimization for E. coli expression

    • Use of specialized expression strains (Rosetta, Arctic Express)

    • Fusion with solubility-enhancing tags (SUMO, MBP)

    • Lower induction temperatures (16-18°C) for extended periods

• Improper Oligomeric Assembly

  • Challenge: Failure to form functional tetradecameric complex

  • Solutions:

    • Optimize buffer conditions with various salts and additives

    • Co-expression with cognate unfoldase partners

    • Addition of peptidic activators during purification

    • Refolding protocols with controlled oligomerization steps

• Protein Instability

  • Challenge: Loss of activity during purification and storage

  • Solutions:

    • Addition of stabilizing agents (glycerol, reducing agents)

    • Finding optimal pH and ionic strength conditions

    • Flash-freezing in liquid nitrogen with cryoprotectants

    • Avoiding multiple freeze-thaw cycles

Functional Analysis Challenges:

• Unfoldase Partner Identification

  • Challenge: Unknown cognate AAA+ unfoldase partners in N. europaea

  • Solutions:

    • Genomic analysis to identify potential ClpA/ClpX homologs

    • Co-immunoprecipitation studies to identify interacting partners

    • Heterologous complementation with E. coli unfoldases

    • Systematic testing of activity with various potential partners

• Substrate Specificity Determination

  • Challenge: Limited knowledge of natural substrates

  • Solutions:

    • Proteomic comparison of wild-type and ClpP-depleted N. europaea

    • Design of peptide libraries to map cleavage preferences

    • Targeted degradation assays with candidate substrates

    • Comparative analysis with ClpP from related bacteria

• Physiological Relevance Assessment

  • Challenge: Connecting in vitro findings to in vivo function

  • Solutions:

    • Generation of conditional ClpP mutants in N. europaea

    • Development of in vivo activity reporters

    • Correlation of in vitro activity with stress response phenotypes

    • Multi-omics approaches to link ClpP activity to cellular physiology

Technical and Methodological Challenges:

• Genetic Manipulation Limitations

  • Challenge: N. europaea is difficult to transform and manipulate genetically

  • Solutions:

    • Optimization of electroporation protocols

    • Development of specialized vectors for N. europaea

    • Use of homologous recombination for chromosomal integration

    • Application of CRISPR-Cas9 systems adapted for N. europaea

• Slow Growth and Culture Challenges

  • Challenge: N. europaea grows slowly as an obligate chemolithoautotroph

  • Solutions:

    • Planning longer experimental timelines

    • Development of more efficient culture methods

    • Use of bioreactors with optimized ammonia supply

    • Careful experimental design to account for growth limitations

• Activity Assay Sensitivity

  • Challenge: Detecting potentially low or specific ClpP activity

  • Solutions:

    • Development of high-sensitivity fluorogenic substrates

    • Use of coupled enzyme assays to amplify signal

    • Application of advanced proteomics methods for substrate tracking

    • Single-molecule approaches for mechanistic studies

By systematically addressing these challenges with the proposed solutions, researchers can overcome the difficulties associated with studying N. europaea ClpP and make significant advances in understanding its role in this environmentally important bacterium .

How can recombinant N. europaea ClpP be utilized for biotechnological applications?

Recombinant Nitrosomonas europaea ClpP offers several promising avenues for biotechnological applications, leveraging its proteolytic activity and the unique ecological niche of its host organism:

Environmental Monitoring and Biosensing:

Bioremediation Enhancement:

• Engineered N. europaea Strains

  • Overexpression of optimized ClpP to enhance stress tolerance

  • Engineering of ClpP substrate specificity for improved degradation of specific toxicants

  • Development of strains with enhanced proteostasis for bioremediation of chlorinated compounds

  • Co-expression of ClpP with other stress-response factors for robust environmental applications

• Immobilized Cell Technologies

  • Development of immobilized N. europaea with engineered ClpP for continuous bioremediation

  • Enhanced stability and longevity of immobilized cells through improved protein quality control

  • Biofilm-based systems with optimized ClpP expression for wastewater treatment

Protein Engineering Applications:

• Controllable Proteolysis Systems

  • Development of regulated proteolytic modules based on N. europaea ClpP

  • Creation of synthetic degradation tags for temporal control of protein function

  • Engineered ClpP variants with altered specificity for targeted protein degradation

  • Temperature or chemical-sensitive variants for controlled proteolysis

• Industrial Enzyme Applications

  • Engineering stress-resistant variants for industrial processes

  • Development of ClpP-based enzyme systems for specific proteolytic applications

  • Creation of chimeric enzymes combining ClpP with other functional domains

Table 7: Potential Biotechnological Applications of Recombinant N. europaea ClpP

Implementation Strategy:

• Laboratory Development Phase

  • Characterization of N. europaea ClpP structure and function

  • Protein engineering to optimize desired properties

  • Small-scale testing of applications in controlled conditions

• Scale-Up and Validation

  • Pilot studies for environmental applications

  • Optimization of expression systems for larger-scale production

  • Field testing of biosensor applications

• Integration with Existing Technologies

  • Combination with established wastewater treatment processes

  • Integration with environmental monitoring networks

  • Development of standardized protocols for industrial applications

The unique properties of N. europaea as an ammonia oxidizer, combined with the central role of ClpP in stress response and proteostasis, create opportunities for innovative biotechnological applications that leverage both the specificity of this enzyme and the ecological significance of its host organism .

What are the key research papers for scientists beginning work on N. europaea ClpP?

For scientists beginning work on Nitrosomonas europaea ClpP, the following key research papers provide essential background, methodological approaches, and contextual understanding:

Foundational Literature on N. europaea:

• Chain et al. (2003). "Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea."

  • Provides the complete genomic sequence and annotation of N. europaea

  • Essential reference for gene identification and genomic context

  • Insights into the unique metabolism of this ammonia-oxidizing bacterium

• Stein et al. (2007). "Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas europaea."

  • Comprehensive analysis of metabolic pathways and gene regulation

  • Important for understanding the cellular context in which ClpP functions

  • Review of stress response pathways relevant to ClpP function

ClpP Structure and Function:

• Bhandari et al. (2018). "ClpP Protease, a Promising Antimicrobial Target."

  • Comprehensive review of ClpP structure, function, and regulation

  • Discussion of ClpP as a potential therapeutic target

  • Overview of compounds that modulate ClpP activity

• Gersch et al. (2012). "AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control."

  • Mechanistic insights into ClpP activation by AAA+ chaperones

  • Foundational for understanding ClpP regulation

  • Methodological approaches for studying ClpP activation

• Kang et al. (2004). "Crystal structure of ClpP from Streptococcus pneumoniae."

  • High-resolution structure of a bacterial ClpP

  • Structural basis for understanding ClpP function

  • Model for homology modeling of N. europaea ClpP

N. europaea Stress Response and Gene Expression:

• Gvakharia et al. (2007). "Global transcriptional analysis of Nitrosomonas europaea to chloroform and chloromethane."

  • Analysis of gene expression changes in response to chlorinated compounds

  • Identification of stress response genes including Clp family members

  • Methodological approaches for studying gene expression in N. europaea

• Radniecki et al. (2009). "Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform or chloromethane."

  • Demonstrates the use of promoter-GFP fusions in N. europaea

  • Relevant to developing ClpP-based biosensors

  • Technical protocols for genetic manipulation of N. europaea

Methodological Papers:

• Frees et al. (2007). "Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria."

  • Protocols for studying Clp proteases in bacteria

  • Methods for substrate identification and activity assays

  • Approaches for studying ClpP function in vivo

• Feng et al. (2013). "Global analysis of protein degradation in Bacillus subtilis."

  • Quantitative proteomics approaches for identifying protease substrates

  • Technical framework adaptable to N. europaea studies

  • Methods for measuring protein half-lives in bacteria

• Hackl et al. (2015). "Isotopically labeled proteome as an internal standard for multiple reaction monitoring-based biomarker quantification."

  • Advanced proteomics methods applicable to ClpP substrate identification

  • Quantitative approaches for measuring protein abundance changes

  • Technical considerations for proteomics in challenging bacterial systems

These key papers collectively provide the theoretical foundation, methodological approaches, and contextual understanding necessary for beginning research on N. europaea ClpP. Scientists should start with the genome paper and ClpP review to build foundational knowledge before exploring more specialized aspects of the research area .

What reagents and resources are most valuable for researchers working with recombinant N. europaea ClpP?

Researchers working with recombinant Nitrosomonas europaea ClpP will benefit from access to the following reagents, materials, and resources:

Biological Materials:

• Bacterial Strains

  • Nitrosomonas europaea ATCC 19718 (reference strain)

  • E. coli expression strains optimized for difficult proteins:

    • BL21(DE3) Star: Enhanced mRNA stability

    • Rosetta(DE3): Contains rare tRNAs for improved expression

    • Arctic Express: Cold-adapted chaperones for improved folding

    • SHuffle: Enhanced disulfide bond formation

• Genetic Constructs

  • Codon-optimized N. europaea clpP gene

  • Expression vectors with various tags:

    • pET28a: N/C-terminal His6-tag options

    • pMAL-c5X: MBP fusion for enhanced solubility

    • pGEX: GST fusion for alternative purification

    • pSUMO: SUMO fusion for enhanced solubility and tag removal

• Related Proteins

  • Purified AAA+ unfoldases (ClpX, ClpA) for activity reconstitution

  • Inactive ClpP variants (S97A) for substrate trapping

  • Fluorescently labeled model substrates (GFP-ssrA, FITC-casein)

Chemical Reagents:

• Activity Assays

  • Fluorogenic peptide substrates (Suc-LY-AMC, Boc-LRR-AMC)

  • ATP and ATP regeneration system components

  • Protease inhibitor panels (for selectivity testing)

  • Activity-based probes specific for serine proteases

• Protein Purification

  • High-quality affinity resins (Ni-NTA, amylose, glutathione)

  • Size exclusion chromatography matrices (Superdex 200)

  • Ion exchange resins (Q-Sepharose, SP-Sepharose)

  • Protease inhibitor cocktails (without serine protease inhibitors)

• Protein Analysis

  • Anti-His tag antibodies for Western blotting

  • Custom anti-ClpP antibodies (if available)

  • Protein crosslinking reagents (BS3, DSS, formaldehyde)

  • Mass spectrometry-grade trypsin and chymotrypsin

Technical Equipment:

• Protein Purification

  • FPLC/HPLC system for automated purification

  • Specialized columns for oligomeric proteins

  • Ultracentrifuge for sedimentation analysis

  • Tangential flow filtration for large-scale preparation

• Protein Characterization

  • Circular dichroism spectrometer for secondary structure analysis

  • Dynamic light scattering for oligomeric state determination

  • Isothermal titration calorimetry for binding studies

  • Differential scanning fluorimetry for stability assessment

• Activity Measurement

  • Fluorescence plate reader with temperature control

  • Stopped-flow apparatus for kinetic studies

  • Mass spectrometer for degradation product analysis

  • Gel documentation system for SDS-PAGE analysis

Computational Resources:

• Sequence Analysis

  • N. europaea genome database access

  • BLAST and other sequence alignment tools

  • Protein structure prediction software

  • Molecular dynamics simulation capabilities

• Structural Analysis

  • Homology modeling software (MODELLER, SWISS-MODEL)

  • Molecular visualization tools (PyMOL, Chimera)

  • Ligand docking software for inhibitor studies

  • Molecular dynamics simulation packages

Table 8: Specialized Resources for Different Aspects of N. europaea ClpP Research

Access to these reagents and resources will facilitate efficient research progress and enable comprehensive characterization of N. europaea ClpP structure, function, and regulation .

What online databases and computational tools are most useful for analyzing N. europaea ClpP?

Researchers studying Nitrosomonas europaea ClpP can leverage numerous online databases and computational tools to enhance their analyses. These resources range from genome browsers to specialized protein analysis platforms and can significantly accelerate research progress:

Genome and Sequence Databases:

• GenBank/NCBI

  • Access to complete N. europaea genome sequence (accession NC_004757)

  • BLAST searches for homology identification

  • Gene neighborhood analysis for genomic context

  • URL: https://www.ncbi.nlm.nih.gov/

• UniProt

  • Curated protein information for ClpP (Q82T33)

  • Functional annotations and sequence features

  • Cross-references to other databases

  • URL: https://www.uniprot.org/

• IMG/JGI

  • Integrated Microbial Genomes database

  • Comparative genomics tools

  • Metabolic reconstruction and pathway analysis

  • URL: https://img.jgi.doe.gov/

Protein Structure Analysis:

• PDB (Protein Data Bank)

  • Repository of solved protein structures

  • ClpP structures from related organisms (templates for homology modeling)

  • Visualization tools and structural analysis

  • URL: https://www.rcsb.org/

• SWISS-MODEL

  • Automated homology modeling server

  • Template identification and quality assessment

  • Interactive model building and refinement

  • URL: https://swissmodel.expasy.org/

• I-TASSER

  • Protein structure and function prediction

  • Integration of threading and ab initio modeling

  • Confidence score estimation for predictions

  • URL: https://zhanggroup.org/I-TASSER/

• AlphaFold DB

  • Access to AlphaFold predicted structures

  • High-quality structure predictions for proteins without experimental structures

  • URL: https://alphafold.ebi.ac.uk/

Functional Analysis Tools:

• Pfam

  • Protein family database

  • Domain identification and annotation

  • Multiple sequence alignments of protein families

  • URL: http://pfam.xfam.org/

• KEGG (Kyoto Encyclopedia of Genes and Genomes)

  • Metabolic pathway mapping

  • Functional hierarchy of genes

  • Cross-species pathway comparison

  • URL: https://www.genome.jp/kegg/

• STRING

  • Protein-protein interaction networks

  • Functional association predictions

  • Integration of experimental and computational data

  • URL: https://string-db.org/

Evolutionary Analysis:

• MUSCLE/Clustal Omega

  • Multiple sequence alignment tools

  • Essential for comparative analysis

  • Identification of conserved residues

  • URLs: https://www.ebi.ac.uk/Tools/msa/muscle/ and https://www.ebi.ac.uk/Tools/msa/clustalo/

• MEGA X

  • Molecular evolutionary genetics analysis

  • Phylogenetic tree construction

  • Selection analysis and evolutionary rate calculation

  • URL: https://www.megasoftware.net/

• ConSurf

  • Evolutionary conservation analysis

  • Mapping conservation scores onto protein structures

  • Identification of functionally important regions

  • URL: https://consurf.tau.ac.il/

Specialized Protease Resources:

• MEROPS

  • Specialized database for proteases

  • Classification and annotation of proteolytic enzymes

  • Substrate specificity information

  • URL: https://www.ebi.ac.uk/merops/

• TopFIND

  • Protein termini and processing database

  • Information on protein cleavage events

  • Substrate-protease relationships

  • URL: https://topfind.clip.msl.ubc.ca/

Table 9: Computational Workflows for Specific N. europaea ClpP Analyses