Recombinant Pseudomonas syringae pv. tomato Thymidylate synthase (thyA)

What is the genomic context of thyA in Pseudomonas syringae pv. tomato DC3000?

Thymidylate synthase (thyA) in P. syringae pv. tomato DC3000 is located on the chromosome at position 6006968-6007939 on the negative strand. The gene is identified by locus tag PSPTO_5282 (previously PSPTO5282) and encodes a protein with a molecular weight of 36.7 kDa and an isoelectric point of 5.60 . Understanding this genomic context is essential for designing appropriate primers for cloning and expression studies. When amplifying thyA, researchers should consider:

• Including 200-300 base pairs upstream of the coding sequence to capture potential regulatory elements

• Designing primers with appropriate restriction sites compatible with your expression vector

• Confirming the sequence integrity after cloning to ensure no mutations were introduced

How can I design an effective expression system for recombinant thyA from P. syringae pv. tomato?

When designing an expression system for recombinant thyA from P. syringae pv. tomato, researchers should consider:

• Vector selection: pET-based expression systems are commonly used for high-level expression of recombinant proteins in E. coli. For P. syringae proteins, vectors that allow moderate expression are often preferable to avoid inclusion body formation.

• Affinity tags: Consider adding an N- or C-terminal His-tag for purification. Based on the charge properties of thyA (charge at pH 7: -6.96), placing the tag at the N-terminus may be advantageous to avoid interfering with the active site .

• Expression host: BL21(DE3) or its derivatives are recommended for recombinant expression. For P. syringae proteins, lower expression temperatures (16-20°C) often improve solubility.

• Codon optimization: Although not always necessary, codon optimization for E. coli expression may improve yields for some P. syringae proteins.

A methodology similar to that used for virulence factors in P. syringae could be adapted, where primers with appropriate restriction sites (e.g., BamHI, PstI) are used to amplify the target gene for subsequent cloning into expression vectors .

What are the key structural and functional characteristics of thyA that affect its recombinant expression?

ThyA (thymidylate synthase) from P. syringae pv. tomato has several characteristics that researchers should consider:

• Hydrophobicity: With a Kyte-Doolittle hydrophobicity value of -0.308, thyA is moderately hydrophilic, which generally favors soluble expression .

• Charge properties: Its negative charge (-6.96) at physiological pH suggests good solubility in standard buffer systems .

• Conserved domains: As a thymidylate synthase, it contains highly conserved domains for substrate binding and catalysis that should be preserved during recombinant expression.

• Oligomeric state: Thymidylate synthases typically function as dimers, which may affect purification and activity assessment protocols.

These characteristics should guide your expression strategy, including buffer composition and purification approach. When designing activity assays, remember that thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP using methylenetetrahydrofolate as a cofactor.

How can I assess if my recombinant thyA retains proper folding and enzymatic function?

To assess proper folding and enzymatic function of recombinant thyA from P. syringae pv. tomato:

• Spectroscopic methods:

  • Circular dichroism (CD) to evaluate secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • Thermal shift assays to evaluate protein stability

• Enzymatic activity assay:

  • Measure conversion of dUMP to dTMP using spectrophotometric methods

  • Monitor consumption of methylenetetrahydrofolate (CH2H4folate)

  • Compare kinetic parameters (Km, kcat) with published values for bacterial thymidylate synthases

• Functional complementation:

  • Test whether recombinant thyA can complement a thyA deficient strain

  • This approach provides physiologically relevant confirmation of activity

A comparative approach using wild-type and mutant variants can provide insights into structure-function relationships. For instance, using methodologies similar to those employed for characterizing virulence factors in P. syringae, you could express the protein in a thyA-deficient strain to assess functional complementation .

How does thyA expression correlate with virulence in P. syringae pv. tomato, and can recombinant thyA be used to study this relationship?

While thyA itself has not been directly identified as a virulence factor in the provided search results, its role in DNA synthesis makes it essential for bacterial growth and potentially important during infection. To study potential correlations:

• Gene expression analysis:

  • Compare thyA expression levels between in vitro growth and in planta conditions

  • Analyze expression in relation to known virulence genes like those regulated by HrpL

  • Use qRT-PCR or RNA-seq approaches similar to those used for studying virulence genes like tvrR

• Functional studies with recombinant protein:

  • Develop thyA knock-out or conditional mutants

  • Complement with recombinant wild-type or modified thyA variants

  • Assess impact on bacterial growth in planta and disease symptom development

• Interaction studies:

  • Investigate potential interactions with known virulence proteins

  • Examine if thyA activity is modulated during infection

While not directly associated with the type III secretion system or coronatine production (key virulence mechanisms in P. syringae), thyA function could be critical during the rapid bacterial multiplication phase within plant tissue .

What methodological approaches can be used to study the interaction between recombinant thyA and plant defense responses?

Given that P. syringae pv. tomato interacts with plant defense mechanisms, studying how thyA might be involved requires specialized approaches:

• Purified protein infiltration studies:

  • Infiltrate purified recombinant thyA into plant leaf tissue

  • Monitor defense responses (ROS production, callose deposition, PR gene expression)

  • Compare responses to known PAMPs (pathogen-associated molecular patterns)

• Protein-protein interaction studies:

  • Use pull-down assays with plant extracts to identify potential plant targets

  • Employ yeast two-hybrid or BiFC (Bimolecular Fluorescence Complementation) to validate interactions

  • Investigate if thyA interacts with plant nucleotide metabolism enzymes

• Metabolite analysis:

  • Monitor changes in nucleotide pools in plant tissue upon infection

  • Compare wild-type infection with thyA mutant infection

  • Assess if thyA activity affects levels of defense-related metabolites

Similar to studies of chemoreceptors like PsPto-PscC that perceive plant signals during infection , thyA might be involved in adaptation to the plant environment through its role in DNA metabolism.

How can I design a high-throughput screening approach to identify inhibitors of recombinant P. syringae thyA with potential for plant disease management?

A structured approach for inhibitor screening would include:

• Assay development:

  • Optimize a spectrophotometric or fluorescence-based activity assay

  • Adapt to 96 or 384-well format

  • Establish Z-factor and signal-to-background ratios for quality control

• Primary screening protocol:

  • Screen compound libraries at a fixed concentration (typically 10-50 μM)

  • Include appropriate positive controls (known thyA inhibitors like 5-fluorouracil)

  • Define hit criteria (typically >50% inhibition)

• Secondary screening and validation:

  • Determine IC50 values for hit compounds

  • Test for selectivity against bacterial vs. plant or human thyA

  • Evaluate compounds for antimicrobial activity against P. syringae

• In planta validation:

  • Test top inhibitors for disease control in plant infection assays

  • Monitor bacterial growth in planta in presence of inhibitors

  • Assess plant toxicity and systemic movement of compounds

This approach parallels methods used to identify compounds targeting other essential bacterial enzymes while being distinct from studies focusing on virulence factors like the type III secretion system .

What are the critical factors for optimizing the solubility of recombinant thyA during heterologous expression?

Optimizing solubility of recombinant thyA requires attention to several key factors:

• Expression temperature and induction conditions:

  • Lower temperatures (16-20°C) often increase solubility

  • Gradual induction with lower IPTG concentrations (0.1-0.5 mM)

  • Extended expression periods (16-24 hours) at lower temperatures

• Buffer optimization:

  • Based on thyA's isoelectric point (pI = 5.60), buffers with pH 7.5-8.0 are recommended

  • Include stabilizing agents like glycerol (5-10%)

  • Test various salt concentrations (100-500 mM NaCl)

• Fusion partners to enhance solubility:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

• Co-expression with chaperones:

  • GroEL/GroES system

  • DnaK/DnaJ/GrpE system

A systematic approach testing combinations of these factors should be implemented. For example, comparing expression at 37°C for 4 hours versus 18°C for 18 hours with varying IPTG concentrations would provide valuable optimization data. A similar methodological approach was used for expressing virulence factors from P. syringae as described in the literature .

How can isotope labeling be optimized for structural studies of recombinant thyA from P. syringae pv. tomato?

For NMR and other structural studies requiring isotope labeling:

• Minimal media composition for isotope labeling:

  • Base medium: Similar to HDM medium used for P. syringae culture (50 mM potassium phosphate, pH 5.7, 1.7 mM sodium chloride, 7.6 mM ammonium sulfate, and 1.7 mM magnesium chloride)

  • Carbon source: 13C-glucose (2-4 g/L)

  • Nitrogen source: 15NH4Cl (1 g/L)

  • Trace elements and vitamins

• Expression optimization in minimal media:

  • Pre-adaptation of cells to minimal media before induction

  • Extended induction times (16-24 hours)

  • Lower temperature (18°C) to compensate for slower growth

• Selective labeling strategies:

  • Amino acid type-selective labeling for specific NMR experiments

  • Deuteration protocols for larger proteins (>20 kDa)

• Purification considerations:

  • Maintain protein stability during extended purification

  • Minimize number of purification steps to preserve yield

  • Buffer optimization for NMR experiments

A typical protocol might involve growing cells in rich media until OD600 ~0.8, then gentle centrifugation and resuspension in isotopically labeled minimal media, followed by a 30-minute adaptation period before induction.

What strategies can address complications arising from thyA's role in nucleotide metabolism when creating knockout strains for complementation studies?

Creating thyA knockout strains presents unique challenges due to its essential role in thymidine synthesis:

• Supplementation strategies:

  • Provide thymidine (50-100 μg/ml) in growth media

  • Use rich media with yeast extract containing thymidine precursors

  • Consider concentration gradients to determine optimal supplementation

• Conditional knockout approaches:

  • Temperature-sensitive mutants

  • Controllable promoter systems (like PBAD or Ptet)

  • Antisense RNA strategies

• Complementation testing:

  • Plasmid-based expression with inducible promoters

  • Integration of recombinant thyA into alternative genomic locations

  • Co-expression of wild-type and mutant thyA to assess dominant-negative effects

• Growth analysis protocol:

  • Monitor growth curves with and without thymidine

  • Assess viability using Live/Dead staining

  • Examine morphological changes by microscopy

Such approaches have been successful for studying other essential genes in P. syringae. A systematic testing of thyA functionality could employ methods similar to those used for virulence factors like tvrR, adapting the complementation strategies to account for thyA's essential nature .

How should kinetic data for recombinant thyA be analyzed to account for bacterial-specific regulatory mechanisms?

Proper analysis of thyA kinetic data requires consideration of P. syringae-specific regulatory factors:

• Steady-state kinetics analysis:

  • Determine Km for dUMP and methylenetetrahydrofolate

  • Calculate kcat and catalytic efficiency (kcat/Km)

  • Compare with parameters from other bacterial species

• Allosteric regulation assessment:

  • Test potential allosteric effectors (dTTP, folate derivatives)

  • Generate Hill plots to identify cooperative binding

  • Analyze data using appropriate non-Michaelis-Menten models if needed

• pH and temperature dependencies:

  • Determine pH optima relevant to P. syringae's natural environment (pH 5.5-6.5 in plant apoplast)

  • Analyze temperature effects reflecting plant infection conditions (18-28°C)

This analytical approach aligns with methods used to study enzyme activities in P. syringae during infection processes, as seen in studies of metabolic adaptations during plant colonization .

What bioinformatic approaches can identify unique structural features of P. syringae thyA compared to plant homologs for selective inhibitor design?

To identify unique features for selective inhibitor design:

• Comparative sequence analysis:

  • Multiple sequence alignment of thyA from P. syringae, plants, and humans

  • Identification of conserved catalytic residues versus divergent regions

  • Analysis of Pseudomonas-specific sequence motifs

• Homology modeling and structural comparison:

  • Generate homology model based on crystal structures of bacterial thyA

  • Compare with plant thyA structures

  • Identify differential binding pocket features and surface properties

• Molecular dynamics simulations:

  • Analyze dynamics of substrate binding sites

  • Identify transiently open pockets unique to bacterial enzymes

  • Evaluate water networks and flexibility differences

• Virtual screening workflow:

  • Define bacterial-specific binding sites

  • Screen compound libraries against P. syringae thyA model

  • Counter-screen against plant thyA models to prioritize selective compounds

This approach parallels methods used in studying species-specific features of other enzymes in plant pathogens, while focusing on the unique aspects of nucleotide metabolism in P. syringae .

How can contradictory results in thyA activity be reconciled when comparing in vitro versus in planta experimental conditions?

Contradictory results between in vitro and in planta conditions are common and can be addressed through:

• Environmental factors assessment:

  • Compare buffer conditions to apoplastic fluid composition

  • Adjust pH to match plant environment (pH 5.5-6.5)

  • Include plant metabolites that may affect enzyme activity

• Post-translational modifications:

  • Assess phosphorylation or other modifications occurring in planta

  • Compare enzyme from cultured bacteria versus bacteria isolated from plants

  • Use phosphomimetic mutations to test effects of potential modifications

• Protein-protein interactions:

  • Test if plant proteins interact with and modify thyA activity

  • Investigate if bacterial virulence factors affect thyA function

  • Use pull-down experiments with plant extracts to identify interactors

• Methodological reconciliation approach:

  • Systematically vary conditions to bridge in vitro and in planta observations

  • Develop intermediate complexity assays (e.g., using apoplastic fluid extracts)

  • Consider time-dependent changes occurring during infection

This approach resembles methods used to study bacterial adaptation to plant environments, as seen in research on chemoreceptors that respond to plant-derived signals like GABA and L-Pro during the infection process .

How can recombinant thyA be utilized to study bacterial adaptation to plant-derived antimicrobial compounds?

Recombinant thyA can serve as a model system for studying bacterial adaptations:

• Resistance mechanism studies:

  • Express thyA variants from resistant strains

  • Characterize kinetic parameters and inhibitor binding

  • Correlate structural changes with resistance phenotypes

• Adaptation to plant-derived inhibitors:

  • Test thyA activity in presence of plant secondary metabolites

  • Identify compounds that interact specifically with bacterial thyA

  • Study evolutionary adaptations in thyA across P. syringae pathovars

• Experimental evolution approach:

  • Generate adapted strains through serial passage with sublethal inhibitor concentrations

  • Sequence thyA and identify mutations

  • Express and characterize recombinant mutant proteins

• Differential susceptibility analysis:

  • Compare thyA from different P. syringae pathovars

  • Correlate variations with host range and infection strategies

  • Identify host-specific adaptations in enzyme function

This application connects to research on bacterial adaptation mechanisms during plant infection, similar to studies of chemoreceptors that evolve to detect specific plant-derived compounds during pathogenesis .

What methodological considerations are critical when using recombinant thyA as a target for structure-based antimicrobial development?

Structure-based antimicrobial development targeting thyA requires:

• High-resolution structural data acquisition:

  • X-ray crystallography of P. syringae thyA with substrates/inhibitors

  • Cryo-EM for capturing different conformational states

  • NMR for dynamics and ligand binding studies

• Fragment-based drug design workflow:

  • Screen fragment libraries against purified thyA

  • Use thermal shift assays, STD-NMR, or crystallography for fragment validation

  • Employ fragment growing, merging, and linking strategies

• Computational screening optimization:

  • Generate P. syringae-specific pharmacophore models

  • Implement molecular dynamics-based virtual screening

  • Use quantum mechanics calculations for transition state inhibitors

• Resistance prediction and mitigation:

  • Identify resistance hotspots through sequence analysis

  • Design inhibitors targeting highly conserved residues

  • Develop combination approaches targeting multiple sites

This structured approach reflects advanced drug discovery methods while focusing specifically on the unique aspects of targeting thyA in the context of plant pathology, distinct from commercial applications.

How can CRISPR-Cas9 technologies be optimized for studying thyA function in P. syringae pv. tomato?

CRISPR-Cas9 approaches for studying thyA require specialized considerations:

• CRISPR system adaptation for P. syringae:

  • Optimize codon usage of Cas9 for Pseudomonas expression

  • Select appropriate promoters (e.g., constitutive vs. inducible)

  • Engineer efficient guide RNA delivery systems

• Guide RNA design strategy:

  • Design gRNAs targeting non-essential regions of thyA for functional studies

  • Create libraries of gRNAs for domain-specific targeting

  • Implement off-target prediction algorithms specific to P. syringae genome

• Homology-directed repair templates:

  • Design templates for precise mutations in catalytic residues

  • Create reporter fusions for localization studies

  • Develop conditional alleles for essential function investigation

• Phenotypic characterization protocol:

  • Assess growth in minimal versus supplemented media

  • Evaluate virulence in susceptible plant hosts

  • Monitor nucleotide pool balance and stress responses

This methodological approach builds upon emerging genome editing technologies adapted specifically for studying essential genes in plant pathogens, representing an advancement over traditional mutagenesis methods used in earlier studies of P. syringae virulence factors .