Recombinant Pseudomonas syringae pv. tomato Chaperone protein ClpB (clpB), partial

What is the primary function of ClpB protein in Pseudomonas syringae pv. tomato?

ClpB functions as an AAA+ ATPase that provides energy for protein secretion via ATP hydrolysis in the bacterial Type VI Secretion System (T6SS) . In P. syringae, ClpB (sometimes named ClpV in P. aeruginosa genome) constitutes a critical component of the secretion machinery that contributes to bacterial virulence during plant infection. Unlike conventional chaperones that primarily assist in protein folding, the ClpB in P. syringae appears specialized for energizing the secretion apparatus, enabling the translocation of effector proteins into host cells. This function places ClpB at the center of pathogenicity mechanisms in this important plant pathogen.

How does ClpB contribute to bacterial virulence mechanisms?

ClpB contributes to virulence through its essential role in the T6SS, which has been revealed as a functional secretion system in P. syringae . The protein enables secretion of Hcp and potentially other virulence factors that facilitate bacterial colonization and infection of plant tissues. Mutation studies have demonstrated that disruption of ClpV (the ClpB homolog) in P. syringae pv. syringae B728a impairs T6SS function, affecting multiple virulence-associated phenotypes including plant colonization . This indicates that the ATPase activity of ClpB is integral to the bacterium's ability to establish successful infections in host plants.

What experimental approaches are most effective for detecting ClpB expression?

Several methodological approaches have proven effective for studying ClpB expression:

• Western blot analysis: Using antibodies specific to ClpB to detect its presence in bacterial lysates and secreted fractions

• Quantitative RT-PCR: Monitoring clpB transcript levels under different environmental conditions or in various mutant backgrounds

• Reporter gene fusions: Constructing transcriptional or translational fusions between the clpB promoter and reporter genes like lacZ or GFP

• RNA-seq analysis: Examining global transcriptional changes to identify conditions affecting clpB expression

These methods can be employed in combination to provide a comprehensive understanding of ClpB expression patterns during infection processes.

What is the relationship between ClpB and the Type VI Secretion System?

ClpB serves as a crucial energizing component of the T6SS, which has been identified as a 29.9-kb gene cluster in the P. syringae pv. syringae B728a genome . Western blot analyses have confirmed that P. syringae secretes Hcp, a T6SS hallmark protein, and this secretion is dependent on ClpV (ClpB homolog) . The T6SS represents a sophisticated protein delivery system that translocates effector proteins directly into other cells. As an AAA+ ATPase, ClpB provides the mechanical force required for the assembly, disassembly, and function of the secretion apparatus through ATP hydrolysis, making it indispensable for T6SS operation.

How do regulatory networks control ClpB expression during plant infection?

ClpB expression appears to be under complex regulatory control involving multiple sensor kinase systems. Research has demonstrated that the RetS and LadS sensor kinases reciprocally regulate the T6SS in P. syringae pv. syringae B728a . Additionally, the GacS/GacA two-component system, which is essential for virulence in many plant pathogenic bacteria, influences the expression of numerous virulence factors . The regulation of T6SS components, including ClpB, is likely integrated into these broader signaling networks that respond to environmental cues during the infection process. This regulatory architecture allows the bacterium to coordinate the expression of virulence factors according to specific stages of infection.

How does ClpB interact with other components of the bacterial stress response system?

ClpB likely participates in complex protein-protein interaction networks beyond its role in the T6SS. As a member of the Clp/Hsp100 family of proteins, ClpB typically functions in conjunction with the DnaK-DnaJ-GrpE chaperone system to resolve protein aggregates under stress conditions. In P. syringae, this interaction network may be particularly important during temperature fluctuations on leaf surfaces or exposure to plant defense compounds. Understanding these interaction networks provides insight into how ClpB contributes to bacterial survival under the variable conditions encountered during plant colonization and infection.

What is the relationship between ClpB activity and other virulence factors in Pseudomonas syringae?

Research indicates that ClpB operates within an integrated virulence network. The RetS and LadS sensor kinases that regulate T6SS also modulate several other virulence-related activities, including swarming motility, exopolysaccharide production, and Type III secretion system (T3SS) gene expression . This suggests coordinated regulation of multiple virulence mechanisms during infection. The table below summarizes known interactions between these regulatory systems and various virulence factors:

What expression systems are optimal for producing recombinant P. syringae ClpB protein?

For optimal expression of recombinant P. syringae ClpB protein, researchers should consider:

• Expression vector selection: pET-based vectors with T7 promoter systems often yield high expression levels for bacterial proteins

• Host strain optimization: E. coli BL21(DE3) derivatives, particularly those with enhanced rare codon usage or chaperone co-expression

• Induction conditions: Lower temperatures (16-20°C) and reduced IPTG concentrations often improve solubility of large ATPases

• Solubility enhancement: Addition of fusion tags (His, MBP, SUMO) can improve protein solubility

• Buffer optimization: Including ATP or non-hydrolyzable ATP analogs in purification buffers often stabilizes AAA+ ATPases

Careful optimization of these parameters is essential for obtaining sufficient quantities of active protein for biochemical and structural studies.

What methods effectively measure ClpB ATPase activity and inhibition?

Researchers can employ several methodologies to quantify ClpB ATPase activity:

• Malachite green phosphate assay: Detects inorganic phosphate released during ATP hydrolysis

• Coupled enzymatic assays: Links ATP hydrolysis to NADH oxidation, which can be monitored spectrophotometrically

• Radioactive assays: Using [γ-32P]-ATP to track phosphate release with high sensitivity

• Luciferase-based ATP consumption assays: Measures remaining ATP after incubation with ClpB

For inhibition studies, researchers should:

• Establish dose-response curves with potential inhibitors

• Determine the mechanism of inhibition (competitive, non-competitive, uncompetitive)

• Validate inhibition in bacterial cultures by measuring T6SS function

What plant infection models best demonstrate ClpB's role in pathogenesis?

Several experimental models are suitable for investigating ClpB's contribution to P. syringae pathogenesis:

• Tomato plant model: As the natural host for P. syringae pv. tomato, tomato plants provide the most physiologically relevant system

• Arabidopsis thaliana: Offers genetic tractability and numerous mutant lines for studying plant-pathogen interactions

• Bean plants: Used for P. syringae pv. syringae studies and allows comparative analysis across pathovars

Experimental approaches include:

• Spray inoculation to assess natural infection processes requiring bacterial entry

• Direct infiltration to bypass entry requirements and focus on in planta growth

• Measurement of bacterial population dynamics in planta

• Assessment of disease symptom development over time

• Microscopy to visualize bacterial colonization patterns

How can researchers create and validate ClpB mutants for functional studies?

Effective strategies for ClpB mutational analysis include:

• Targeted mutagenesis approaches:

  • Site-directed mutagenesis of conserved Walker A and B motifs essential for ATP binding and hydrolysis

  • Domain deletion constructs to assess the contribution of individual protein domains

  • Chimeric proteins combining domains from different species to identify host-specific adaptations

• Validation methodologies:

  • Complementation assays to verify phenotype restoration with wild-type clpB

  • Western blot analysis to confirm Hcp secretion as a T6SS function readout

  • ATP hydrolysis assays to directly measure enzymatic activity

  • Plant infection assays to assess virulence in different mutant backgrounds

How should researchers analyze gene expression data for ClpB and related T6SS components?

Robust data analysis for ClpB expression studies should incorporate:

• Normalization strategies:

  • Use multiple reference genes with proven stability under experimental conditions

  • Apply appropriate normalization methods for RNA-seq data (e.g., TPM, RPKM, or DESeq2 normalization)

• Statistical approaches:

  • ANOVA for multi-factor experimental designs

  • Time-series analysis for infection progression studies

  • Correlation analyses to identify co-regulated genes

• Integration with other datasets:

  • Combine transcriptomic data with proteomic analyses

  • Correlate expression with phenotypic measurements

  • Compare expression patterns across different P. syringae pathovars

What computational approaches help predict ClpB structure-function relationships?

Several computational methods provide valuable insights into ClpB structure-function relationships:

• Homology modeling: Using crystal structures of related AAA+ ATPases as templates

• Molecular dynamics simulations: To predict conformational changes during ATP binding and hydrolysis

• Protein-protein docking: To model interactions with other T6SS components

• Conservation analysis: Identifying evolutionarily conserved residues crucial for function

• Coevolution analysis: Detecting co-evolving residue pairs that might indicate functional interactions

These approaches are particularly valuable when combined with experimental validation through site-directed mutagenesis and functional assays.

How can researchers distinguish between direct and indirect effects of ClpB on virulence?

Distinguishing direct from indirect effects requires careful experimental design:

• Genetic approaches:

  • Point mutations affecting specific functions (e.g., ATPase activity) without disrupting protein interactions

  • Conditional expression systems to control timing and level of ClpB expression

  • Epistasis analysis with mutations in other T6SS components

• Biochemical approaches:

  • In vitro reconstitution of T6SS subassemblies to define direct interactions

  • Pull-down assays to identify direct binding partners

  • Time-resolved analyses to establish causality in signaling cascades

• In planta approaches:

  • Microscopy to track localization of fluorescently tagged ClpB during infection

  • Comparative transcriptomics/proteomics between wild-type and clpB mutants at different infection stages

What bioinformatic strategies help identify conserved domains and functional motifs in ClpB proteins?

Comprehensive bioinformatic analysis of ClpB should include:

• Sequence alignment and phylogenetic analysis:

  • Multiple sequence alignment of ClpB homologs across Pseudomonas species

  • Construction of phylogenetic trees to trace evolutionary relationships

  • Identification of pathovar-specific sequence features

• Domain and motif analysis:

  • Identification of canonical AAA+ ATPase domains and Walker A/B motifs

  • Detection of species-specific insertions or deletions

  • Prediction of post-translational modification sites

• Structural bioinformatics:

  • Secondary structure prediction

  • Tertiary structure modeling using tools like AlphaFold

  • Analysis of surface electrostatics to identify potential interaction interfaces

• Genomic context analysis:

  • Examination of operon structure and conservation

  • Identification of regulatory elements in promoter regions

  • Comparative analysis of T6SS gene clusters across species