Recombinant Pseudomonas syringae pv. tomato ATP-dependent Clp protease proteolytic subunit (clpP)

What is the function of ClpP in Pseudomonas syringae pv. tomato?

ClpP is the proteolytic core of the ATP-dependent Clp protease complex in Pseudomonas syringae pv. tomato. This serine protease functions as part of a larger proteolytic system that plays critical roles in protein quality control, stress responses, and virulence regulation. The ClpP subunit forms a tetradecameric barrel-shaped structure composed of two heptameric rings that enclose a central proteolytic chamber. This structure works in conjunction with ATPase components (typically ClpA, ClpX, or ClpC) that recognize, unfold, and translocate substrate proteins into the ClpP proteolytic chamber for degradation.

In P. syringae, as in other bacteria, the ClpP protease system is particularly important during environmental stress conditions and pathogenesis. While ClpP itself hasn't been extensively characterized in P. syringae specifically, its homologs in other pseudomonads regulate important cellular processes including virulence factor production, biofilm formation, and response to oxidative stress.

How can I identify and annotate the clpP gene in newly sequenced P. syringae strains?

To identify and annotate the clpP gene in newly sequenced P. syringae strains, follow this methodological approach:

• Perform BLAST analysis using known ClpP sequences from related Pseudomonas species as queries against your newly sequenced genome.

• Verify the candidate gene using multiple sequence alignment with established ClpP sequences to identify conserved catalytic residues (Ser-His-Asp catalytic triad).

• Analyze the genomic context of the putative clpP gene, as it is often conserved across Pseudomonas species. In most pseudomonads, clpP is found in proximity to genes encoding regulatory proteins or stress response factors.

• Use domain prediction tools (Pfam, InterPro) to confirm the presence of the Peptidase S14 domain characteristic of ClpP proteins.

• Perform phylogenetic analysis to position your ClpP sequence within the evolutionary context of other bacterial ClpP proteins, particularly focusing on other Pseudomonas species.

A typical annotation report should include:

What expression systems are most suitable for producing recombinant P. syringae ClpP?

For expressing recombinant P. syringae ClpP, several expression systems can be utilized, each with distinct advantages:

E. coli expression systems: The most common approach utilizes E. coli BL21(DE3) with pET vector systems. This method typically yields high protein amounts but may produce inclusion bodies requiring refolding. For P. syringae proteins, codon optimization may improve expression, especially when rare codons are present.

Pseudomonas-based expression systems: For more authentic post-translational modifications and proper folding, a homologous expression system using modified P. syringae strains can be advantageous. The pUCP24/47 vector system described in the literature has been successfully used for expressing pseudomonad proteins .

A methodological protocol for ClpP expression in E. coli would include:

• Clone the clpP gene into a suitable expression vector (pET28a with His-tag is commonly used)

• Transform into E. coli BL21(DE3)

• Induce expression with IPTG (typically 0.5 mM) at 25°C overnight to minimize inclusion body formation

• Harvest cells and lyse using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Purify using nickel affinity chromatography followed by size exclusion chromatography

For activity verification, substrate degradation assays using fluorogenic peptides (like Suc-Leu-Tyr-AMC) can confirm proteolytic function of the recombinant protein.

How can I generate a clpP knockout mutant in P. syringae using recombineering?

Generating a clpP knockout mutant in P. syringae can be achieved using recombineering technology as described for P. syringae pv. tomato DC3000. The approach utilizes RecTE homologs identified in P. syringae that promote efficient homologous recombination .

Methodological protocol:

• Construct a knockout cassette with antibiotic resistance marker:

  • Design primers with 50-80 bp homology to regions flanking the clpP gene

  • PCR amplify an antibiotic resistance cassette (e.g., nptII for kanamycin resistance)

  • Verify the construct by sequencing

• Express RecTE recombination proteins:

  • Transform P. syringae with a plasmid expressing RecT or RecTE proteins (e.g., pUCP24/47 with recTE genes)

  • Grow transformants at 28°C in Kings B medium with appropriate antibiotics

• Introduce the knockout construct:

  • Prepare electrocompetent cells from the RecTE-expressing strain

  • Electroporate the knockout cassette (1-2 μg of linear DNA)

  • Outgrow cells for 2-3 hours before plating on selective media

• Screen and verify mutants:

  • Verify correct integration by PCR with primers binding outside the recombination region

  • Confirm clean deletion by sequencing

  • Verify the absence of ClpP protein by Western blot analysis

Recombination frequencies using this system have been reported to range from 10^-3 to 10^-6 recombinants per viable cell, depending on the locus and construct design .

What phenotypic changes might I observe in a P. syringae clpP deletion mutant?

A P. syringae clpP deletion mutant would likely exhibit several observable phenotypic changes:

Growth and stress response alterations:

• Reduced growth rate, particularly under stress conditions

• Increased sensitivity to oxidative stress (H₂O₂ challenge)

• Altered response to heat shock (42°C)

• Impaired growth at low temperatures

• Increased sensitivity to antibiotics targeting protein synthesis

Virulence-related changes:

• Altered production of extracellular polysaccharides (EPS) like alginate and levan, which are important for virulence in P. syringae

• Potential changes in motility (swarming and swimming) that could affect colonization

• Modified biofilm formation capacity

• Altered expression of type III secretion system (T3SS) genes, critical for pathogenicity

Molecular phenotypes:

• Accumulation of misfolded and aggregated proteins

• Changes in the expression of other heat shock proteins (compensatory effect)

• Altered proteomic profile, particularly during stress conditions

Testing methodology should include comparative growth curves under different stress conditions, biofilm formation assays, and plant infection experiments using appropriate host plants (such as tomato or bean) to evaluate changes in pathogenicity.

How does ClpP interact with the Type VI Secretion System (T6SS) in P. syringae?

While direct interactions between ClpP and the Type VI Secretion System (T6SS) in P. syringae have not been explicitly documented in the provided search results, we can infer potential relationships based on known regulatory mechanisms:

The T6SS in P. syringae pv. syringae B728a has been identified as a 29.9-kb gene cluster, and its functionality depends on ClpV, an AAA+ ATPase that is part of the T6SS machinery . While ClpV and ClpP are distinct proteins with different functions, they both play roles in protein quality control and function.

Potential interaction mechanisms include:

• Regulatory role: ClpP may degrade regulatory proteins controlling T6SS expression, such as transcriptional activators or repressors. The RetS/LadS sensor kinase system, which regulates T6SS in P. syringae , might be subject to ClpP-mediated proteolysis.

• Quality control of T6SS components: ClpP likely participates in the quality control of T6SS structural proteins, ensuring proper assembly and function of the secretion apparatus.

• Stress response coordination: Both ClpP and T6SS are involved in stress responses, suggesting potential coordinated regulation wherein ClpP activity modulates T6SS expression or function in response to environmental conditions.

To investigate these interactions experimentally:

• Compare T6SS gene expression and protein levels between wild-type and ΔclpP strains using qRT-PCR and Western blot analysis

• Assess secretion of Hcp (a T6SS-secreted protein) in ΔclpP mutants to determine if ClpP affects T6SS functionality

• Perform co-immunoprecipitation experiments to identify direct protein-protein interactions between ClpP and T6SS components

• Use protein stability assays to determine if regulatory proteins like RetS or LadS are substrates for ClpP-mediated degradation

What purification strategies work best for maintaining the active oligomeric structure of P. syringae ClpP?

Purifying active oligomeric P. syringae ClpP requires careful consideration of conditions that maintain its tetradecameric structure. The following methodological approach is recommended:

Expression and initial purification:

• Express ClpP with a cleavable affinity tag (His₆ tag is preferred) using the pUCP24/47 vector system in P. syringae or E. coli

• Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10% glycerol, 5 mM MgCl₂, and protease inhibitors

• Perform initial purification using nickel affinity chromatography with gradient elution to minimize co-purifying contaminants

• Remove the affinity tag using TEV protease if a cleavage site was incorporated

• Further purify using ion exchange chromatography (typically Q-Sepharose)

Critical factors for maintaining oligomeric structure:

Verification of oligomeric state:

• Size exclusion chromatography (Superose 6 column) to confirm tetradecameric assembly (~300 kDa)

• Native PAGE to assess oligomer integrity

• Dynamic light scattering to measure particle size distribution

• Negative stain electron microscopy to visualize the characteristic barrel-shaped structure

• Activity assays using peptide substrates to confirm functional assembly

For long-term storage, flash-freeze purified ClpP in liquid nitrogen and store at -80°C in small aliquots to avoid repeated freeze-thaw cycles.

How can I identify the substrate specificity of P. syringae ClpP using proteomic approaches?

Identifying ClpP substrates in P. syringae requires sophisticated proteomic approaches that capture the differential protein accumulation when ClpP function is altered. The following methodological workflow is recommended:

1. Generation of substrate trap variants:
Create a proteolytically inactive ClpP variant (typically S97A mutation in the catalytic site) that can still form complexes with its ATPase partners and bind substrates without degrading them. This "trap" variant can be expressed with a His-tag for affinity purification.

2. Comparative proteomics approaches:

a. SILAC (Stable Isotope Labeling with Amino acids in Cell culture):

• Grow wild-type P. syringae in medium with "light" amino acids

• Grow ΔclpP mutant in medium with "heavy" amino acids (e.g., ¹³C₆-Arg, ¹³C₆-Lys)

• Mix cultures 1:1, extract proteins, and analyze by LC-MS/MS

• Proteins with higher abundance in the "heavy" fraction are potential ClpP substrates

b. TMT (Tandem Mass Tag) labeling:

• Extract proteins from wild-type, ΔclpP, and ClpP trap variant strains

• Label peptides with different TMT reagents

• Combine and analyze by LC-MS/MS

• Quantify proteins that accumulate in the absence of ClpP activity

3. In vivo crosslinking coupled with co-immunoprecipitation:

• Treat cells expressing His-tagged ClpP trap variant with formaldehyde

• Lyse cells and perform His-tag pulldown under denaturing conditions

• Identify co-precipitated proteins by LC-MS/MS

4. Validation of candidate substrates:

• Express candidates with epitope tags in wild-type and ΔclpP backgrounds

• Monitor protein stability using cycloheximide chase experiments

• Perform in vitro degradation assays with purified components

5. Bioinformatic analysis of identified substrates:

• Analyze sequence motifs that might serve as degradation signals

• Classify substrates by cellular function and localization

• Compare with known ClpP substrates from other bacteria

This comprehensive approach will yield a proteome-wide view of ClpP substrates in P. syringae and provide insights into its role in cellular physiology and pathogenesis.

How does recombinant ClpP activity differ when expressed in heterologous systems versus native P. syringae?

The functional properties of recombinant P. syringae ClpP can vary significantly depending on the expression system used. Understanding these differences is crucial for accurate biochemical characterization and functional studies.

Key differences between heterologous and native expression:

Methodological approaches to assess differences:

• Side-by-side activity comparison:

  • Purify ClpP from both expression systems under identical conditions

  • Perform peptidase assays using fluorogenic peptides

  • Compare kinetic parameters (Kₘ, Vₘₐₓ, kcat)

• Structural characterization:

  • Analyze oligomeric state using size exclusion chromatography

  • Assess thermal stability using differential scanning fluorimetry

  • Compare circular dichroism spectra to detect secondary structure differences

• Interaction studies with ATPase partners:

  • Purify native P. syringae ClpA/ClpX ATPases

  • Compare binding affinities between heterologous and native ClpP

  • Assess ATP-dependent protein degradation efficiency

• Mass spectrometry analysis:

  • Identify post-translational modifications present in native but not heterologous ClpP

  • Compare protein-protein interaction networks

• Complementation experiments:

  • Test ability of heterologously expressed ClpP to complement a ΔclpP P. syringae strain

These analyses will provide a comprehensive understanding of the functional equivalence (or differences) between heterologously and natively expressed ClpP, informing interpretation of biochemical data and guiding choice of expression system for specific applications.

How can RecTE-based recombineering be optimized for studying ClpP function in P. syringae?

RecTE-based recombineering offers a powerful approach for precise genetic manipulation of P. syringae to study ClpP function. Based on established protocols for P. syringae, the following optimized methodology is recommended :

1. Optimized construction of RecTE expression plasmids:

The RecTE homologs from P. syringae pv. syringae B728a have been shown to function efficiently in P. syringae pv. tomato DC3000 . A two-plasmid system can be developed:

• One plasmid expressing RecT alone for ssDNA recombination

• A second plasmid expressing both RecT and RecE for dsDNA recombination

For optimal expression, use the constitutive BAD promoter (PnptII) as described in the literature, which provides consistent expression levels without the need for induction .

2. Design of genetic modifications for studying ClpP:

3. Protocol optimization for maximum efficiency:

• Grow cells expressing RecTE to mid-log phase (OD₆₀₀ = 0.4-0.6) in Kings B medium

• Prepare highly electrocompetent cells using ice-cold 10% glycerol with multiple washing steps

• Use 100-500 ng of ssDNA oligos or 1-2 μg of dsDNA constructs

• Electroporate at 2.5 kV, 25 μF, 200 Ω

• Recover cells for 3-4 hours in rich media before selection

• For non-selectable changes (point mutations), use MAGE-like approaches with multiple rounds of recombineering

4. Verification strategies:

• For point mutations: RFLP analysis if mutation creates/abolishes restriction site, followed by sequencing

• For insertions/deletions: PCR screening with primers flanking the modified region

• For tagged variants: Western blot using tag-specific antibodies

• For all modifications: Phenotypic verification of expected changes in ClpP function

This optimized RecTE-based recombineering approach achieves recombination frequencies of approximately 10⁻³ for selectable markers and 10⁻⁵-10⁻⁶ for non-selectable modifications , making it highly efficient for studying ClpP function through a variety of genetic manipulations.

What is the role of ClpP in regulating bacterial competition via the Type VI secretion system in P. syringae?

The role of ClpP in regulating bacterial competition through the Type VI secretion system (T6SS) in P. syringae involves complex interactions between proteolytic control mechanisms and secretion machinery. While direct evidence from the search results is limited, research on related systems allows us to construct a model of these interactions.

The T6SS in P. syringae pv. syringae B728a has been identified as a functional secretion system that depends on ClpV (a different AAA+ ATPase than those typically associated with ClpP) . This system mediates antagonistic interactions between bacterial strains, as evidenced by the diverse bacteriocin killing activity documented across P. syringae strains .

Proposed regulatory mechanisms:

• Proteolytic control of T6SS components:
  ClpP likely regulates the abundance of key T6SS structural proteins through targeted degradation, affecting assembly and function of the secretion apparatus.

• Regulation of T6SS expression:
  The RetS/LadS sensor kinase system, which has been shown to regulate T6SS in P. syringae , may be subject to ClpP-mediated proteolytic control. Accumulated data suggests that these sensor kinases reciprocally regulate the T6SS and modulate several virulence-related activities .

• Stress-response coordination:
  ClpP-mediated proteolysis responds to environmental stresses that may also trigger competitive behaviors mediated by the T6SS, suggesting coordinated regulation.

Experimental evidence and methodological approaches:

Analysis of a ΔclpP mutant would likely reveal:

• Altered expression patterns of T6SS genes

• Modified secretion of Hcp (a key T6SS-secreted protein used as a marker of T6SS activity)

• Changes in competitive fitness when co-cultured with other bacterial strains

• Differential regulation of the T6SS in response to environmental stresses

To investigate this systematically:

• Create and characterize a ΔclpP mutant in P. syringae using the RecTE recombineering system

• Perform transcriptomic and proteomic analyses comparing wild-type and ΔclpP strains

• Assess T6SS activity through Hcp secretion assays

• Conduct bacterial competition assays between wild-type and ΔclpP strains against various competitors

• Investigate the stability and abundance of RetS/LadS proteins in the absence of ClpP

The interconnection between proteolytic systems and secretion machinery represents an important regulatory nexus for bacterial competition and environmental adaptation in P. syringae.