Recombinant Pseudomonas syringae pv. tomato UPF0337 protein PSPTO_1596 (PSPTO_1596)

What is the current understanding of PSPTO_1596's function in Pseudomonas syringae pv. tomato?

PSPTO_1596 is classified as a UPF0337 family protein with currently uncharacterized function in Pseudomonas syringae pv. tomato. Based on comparative analyses with other P. syringae pathovars, this protein may be involved in stress response mechanisms or virulence. Research on related P. syringae proteins suggests potential roles in either Type III secretion system (T3SS) or Type VI secretion system (T6SS) pathways, which are critical for pathogenicity and host-microbe interactions . While its precise function remains to be fully characterized, genomic context analysis places it in proximity to genes associated with secretion systems that are activated under apoplast-like conditions.

Methodological approach for functional characterization:

• Perform comparative genomics with characterized proteins in other pathovars

• Generate knockout mutants and assess phenotypic changes in virulence

• Conduct transcriptional analysis under various stress conditions

• Test secretion patterns in apoplast-mimicking media

What growth conditions and media compositions are optimal for expressing recombinant PSPTO_1596?

For optimal expression of recombinant PSPTO_1596, researchers should consider media compositions that mimic relevant physiological conditions:

For recombinant expression, E. coli BL21(DE3) grown in LB medium supplemented with appropriate antibiotics at 37°C until OD₆₀₀ reaches 0.6, followed by induction with IPTG (0.5-1.0 mM) and incubation at 16-18°C overnight has shown good results for various P. syringae proteins. Temperature downshift after induction helps maintain protein solubility.

What purification strategies are most effective for isolating recombinant PSPTO_1596?

The purification of recombinant PSPTO_1596 should follow a systematic approach:

• Expression with affinity tag (6xHis, GST, or MBP) to facilitate purification

• Cell lysis using sonication or French press in buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Primary purification using affinity chromatography:

  • For His-tagged protein: Ni-NTA resin with imidazole gradient elution (20-250 mM)

  • For GST-tagged protein: Glutathione sepharose with reduced glutathione elution

• Secondary purification using size exclusion chromatography:

  • Superdex 75/200 column depending on protein size

  • Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol

• Quality assessment:

  • SDS-PAGE for purity

  • Western blotting for identity confirmation

  • Dynamic light scattering for homogeneity

Modifications for proteins with solubility issues include addition of mild detergents (0.05% Triton X-100) or chaotropic agents at low concentrations during initial purification steps, followed by their removal during size exclusion chromatography.

How can researchers detect the secretion of PSPTO_1596 in bacterial culture supernatants?

Detection of secreted PSPTO_1596 requires careful experimental design:

• Growth conditions optimization:

  • Culture bacteria in hrp-inductive minimal medium (HIM) containing 10 mM fructose at pH 5.7 and 22°C to mimic apoplast conditions

  • Include King's B medium at 28°C as a negative control for non-secreting conditions

• Sample preparation:

  • Culture bacteria to OD₆₀₀ of 0.15-0.2

  • Incubate for 6 hours with shaking (180 rpm)

  • Collect supernatant by centrifugation (5,000-8,000 × g for 10 minutes)

  • Filter through 0.22 μm filter to remove remaining cells

  • Concentrate proteins using TCA precipitation or ultrafiltration

• Detection methods:

  • SDS-PAGE and silver staining for initial visualization

  • Western blotting using antibodies against PSPTO_1596 or its tag

  • Label-free quantitative (LFQ) proteomics for comprehensive secretome analysis

  • Selected reaction monitoring (SRM) mass spectrometry for targeted quantification

• Validation:

  • Electron microscopy to confirm secretion system formation (T3SS pili have a diameter of approximately 7.3 ± 0.8 nm, compared to flagella at 18.0 ± 4.0 nm)

  • Genetic approaches using secretion system mutants to confirm the pathway

How does osmotic stress affect PSPTO_1596 expression and protein secretion patterns?

Osmotic stress significantly impacts Pseudomonas syringae gene expression and protein secretion. Based on studies of P. syringae strains under osmotic upshift conditions:

• Transcriptional responses:

  • Different P. syringae strains show markedly distinct transcriptional responses to osmotic stress

  • Strain B728a primarily shows upregulation of stress response genes, while DC3000 exhibits downregulation of virulence-related genes

  • RNA isolation protocol for transcriptomics:

    • Grow cells to OD₆₀₀ of 0.4 (approximately 8 × 10⁸ cells/ml)

    • Transfer to medium with NaCl (219 mM for -1 MPa osmotic pressure)

    • Incubate for 15 minutes at 28°C with shaking

    • Stabilize RNA with RNAProtect Reagent

    • Extract and quantify RNA for subsequent analysis

• Proteome alterations:

  • Osmotic stress affects the expression and secretion of effector proteins

  • Comparative proteomics reveals distinct secretion patterns:

• Compatible solute accumulation:

  • Some P. syringae strains produce NAGGN (N-acetylglutaminylglutamine amide) to counter osmotic stress

  • The production of exopolysaccharides like alginate increases under osmotic stress conditions

  • Measurement method: Precipitate polysaccharides with cold ethanol (4:1 ratio) and quantify using meta-hydroxydiphenyl assay with D-glucuronic acid as a standard

What techniques are most appropriate for studying potential protein-protein interactions involving PSPTO_1596?

Studying protein-protein interactions involving PSPTO_1596 requires a multi-faceted approach:

• In vitro interaction assays:

  • Pull-down assays using purified recombinant PSPTO_1596 as bait

  • Surface plasmon resonance (SPR) for real-time kinetic studies

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for interaction studies with minimal protein amounts

• In vivo interaction studies:

  • Bacterial two-hybrid (B2H) system

  • Split-GFP complementation assays

  • Co-immunoprecipitation from bacterial lysates

  • Crosslinking mass spectrometry (XL-MS) for capturing transient interactions

• Structural approaches:

  • X-ray crystallography of PSPTO_1596 in complex with binding partners

  • Cryo-electron microscopy for larger complexes

  • NMR spectroscopy for mapping interaction interfaces

• Computational predictions:

  • Molecular docking simulations

  • Coevolutionary analysis to identify potential interaction partners

  • Network analysis of P. syringae protein-protein interaction databases

For secreted proteins like those in the T3SS or T6SS pathways, researchers should also consider plant-pathogen interaction assays to identify potential host targets.

How can researchers resolve contradictory experimental results when studying PSPTO_1596 function?

When faced with contradictory experimental results in PSPTO_1596 research, apply a structured approach similar to proof by contradiction in logic :

• Systematic verification:

  • Carefully document all experimental conditions and variables

  • Repeat experiments with standardized protocols

  • Verify reagent quality and equipment calibration

  • Ensure proper controls for each experiment

• Hypothesis testing framework:

  • Formulate clear, testable hypotheses about PSPTO_1596 function

  • Design experiments to specifically test each hypothesis

  • Apply the principle of proof by contradiction: if assuming a function leads to conflicting results, that function may be incorrect

• Resolution strategies:

  • Cross-validation using multiple independent techniques

  • Collaboration with laboratories with complementary expertise

  • Consider strain-specific effects (as seen in P. syringae strains with different virulence profiles)

  • Temporal analysis to account for expression/activity changes under different conditions

• Statistical analysis:

  • Apply appropriate statistical methods for biological replicates

  • Calculate false discovery rates (FDRs) for high-throughput data

  • Use linear models for microarray data analysis to share information across genes when estimating error variances

  • Consider biological relevance alongside statistical significance

What approaches can be used to characterize the structural properties of PSPTO_1596?

Structural characterization of PSPTO_1596 requires a comprehensive approach:

• Bioinformatics prediction:

  • Secondary structure prediction using PSIPRED or JPred

  • Disorder prediction using PONDR or IUPred

  • Template-based modeling using I-TASSER, Phyre2, or AlphaFold2

  • Functional domain identification using InterProScan

• Experimental structure determination:

  • X-ray crystallography workflow:

    • Optimize protein to >95% purity and stability

    • Screen crystallization conditions (temperature, pH, precipitants)

    • Data collection at synchrotron radiation facility

    • Structure determination and refinement

  • NMR spectroscopy for smaller domains:

    • Isotope labeling (¹⁵N, ¹³C) of recombinant protein

    • Collection of multi-dimensional spectra

    • Assignment of resonances and structure calculation

• Biophysical characterization:

  • Circular dichroism (CD) for secondary structure composition

  • Differential scanning calorimetry (DSC) for thermal stability

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics and conformational changes

• Functional implications:

  • Structure-guided mutagenesis of conserved residues

  • Functional assays to correlate structure with activity

  • Molecular dynamics simulations to understand protein motion

How does PSPTO_1596 contribute to the distinct virulence strategies observed in Pseudomonas syringae pathovars?

Understanding PSPTO_1596's contribution to virulence requires examining the broader secretion system context:

• Current knowledge from related pathovars:

  • P. syringae pathovars exhibit distinct secretion profiles that correlate with virulence capacity

  • Low virulence strains secrete higher levels of most T3SS effectors but have narrower host ranges

  • High virulence strains secrete a distinct subset of four effectors and have broader host ranges

  • T3SS and T6SS activities show potential interplay that shapes pathogenicity strategies

• Research approach for PSPTO_1596:

  • Generate deletion mutants (Δpspto_1596) and complemented strains

  • Assess virulence in planta using standardized infection assays

  • Compare secretome profiles between wild-type and mutant strains

  • Examine transcriptional changes in plant defense genes upon infection

• Experimental design to test contribution:

  • Infect multiple plant species to assess host range impacts

  • Measure disease progression by quantifying necrotic leaf area percentage at 7 days post-inoculation

  • Test mutant survival under various environmental stresses

  • Examine biofilm formation and motility as virulence-related phenotypes

What methodological approaches should be used to study PSPTO_1596 in the context of apoplast colonization?

Studying PSPTO_1596 during apoplast colonization requires specialized methodologies:

• In vitro apoplast simulation:

  • Use hrp-inductive minimal medium (HIM) with 10 mM fructose, pH 5.7 at 22°C to mimic apoplast conditions

  • Compare with non-inducing conditions (King's B medium, 28°C, neutral pH)

  • Monitor protein secretion and expression over time during growth

• In planta studies:

  • Inoculation methods:

    • Syringe infiltration for direct apoplast access

    • Dip or spray inoculation to study natural infection process

  • Bacterial recovery and enumeration from leaf tissue

  • Confocal microscopy with fluorescently tagged PSPTO_1596 to track localization

  • Plant tissue extraction to identify in planta protein interactions

• Transcriptomics approach:

  • RNA isolation from bacteria recovered from apoplast

  • RNA-seq to identify co-regulated genes

  • RT-qPCR validation of expression patterns

  • Comparison with in vitro expression under apoplast-mimicking conditions

• Functional validation:

  • Construct strains with inducible/repressible PSPTO_1596 expression

  • Perform time-course experiments during infection

  • Measure bacterial populations in planta over time

  • Assess plant defense responses using marker genes