Recombinant Pseudomonas syringae pv. tomato Maf-like protein PSPTO_3837 (maf-1)

What is the biochemical classification and fundamental function of Maf-like proteins in Pseudomonas syringae pv. tomato?

Maf-like proteins in P. syringae pv. tomato belong to the broader Maf (multicopy associated filamentation) protein family found across prokaryotes and eukaryotes. The fundamental biochemical activity of Maf proteins has been identified as nucleotide pyrophosphatase activity against both canonical nucleotides (dTTP, UTP, and CTP) and modified nucleotides (5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP) .

These proteins contain a conserved signature motif (S-R-E-K-D-K): Ser10, Arg13, Glu33, Lys52, Asp70, and Lys82, which is essential for their enzymatic function . In bacterial systems, Maf proteins are implicated in cell division processes and potentially in the prevention of incorporation of modified nucleotides into cellular nucleic acids, serving a "house-cleaning" function .

In the context of P. syringae pv. tomato DC3000, Maf-like proteins may participate in regulatory networks involving virulence and pathogenicity, potentially interacting with other regulatory systems such as the PsrA (Pseudomonas sigma regulator) that controls virulence factors .

What expression systems and vectors are optimal for recombinant production of PSPTO_3837?

For optimal recombinant expression of PSPTO_3837, the pET expression system, particularly pET-28a(+), has proven effective for similar bacterial proteins . This approach offers the following advantages:

Expression Protocol:

• Clone the PSPTO_3837 gene using primers designed based on the DC3000 genome sequence

• Insert the gene into pET-28a(+) vector between appropriate restriction sites (typically EcoRI and HindIII)

• Transform the recombinant plasmid into E. coli BL21(DE3) competent cells

• Induce expression using IPTG (typically 0.5-1.0 mM)

• Harvest cells and lyse using appropriate buffer systems

The pET-28a(+) vector provides a His-tag sequence that facilitates subsequent purification using Ni-NTA affinity chromatography . Analysis of the recombinant protein expression should include SDS-PAGE to confirm the expected molecular weight and Western blotting using anti-His antibodies to verify the presence of the fusion protein .

How can researchers verify the structural integrity and enzymatic activity of recombinant PSPTO_3837?

Verification of recombinant PSPTO_3837's structural integrity and enzymatic activity requires a multi-faceted approach:

Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy to analyze secondary structure elements

• Thermal shift assays to determine protein stability

• Dynamic light scattering to assess homogeneity

• Limited proteolysis to identify stable domains

Enzymatic Activity Assays:
The nucleotide pyrophosphatase activity can be assessed using a methodology similar to that employed for other Maf proteins :

The enzymatic reaction can be monitored by measuring the release of pyrophosphate (PPi) using commercially available pyrophosphatase-coupled colorimetric assays or by direct detection of the nucleotide monophosphate products .

What purification strategies yield the highest purity and activity of recombinant PSPTO_3837?

Optimal purification of recombinant PSPTO_3837 with His-tag can be achieved through a sequential purification strategy:

Multi-step Purification Protocol:

• Initial Capture: Ni-NTA affinity chromatography using imidazole gradient elution (typically 20-300 mM)

• Intermediate Purification: Ion exchange chromatography (either anion or cation exchange depending on the protein's pI)

• Polishing Step: Size exclusion chromatography using appropriate buffer conditions (typically 20-50 mM Tris-HCl pH 7.5-8.0, 100-200 mM NaCl, 1-5 mM DTT)

Critical Parameters for Maintaining Activity:

• Include 5-10% glycerol in all buffers to enhance stability

• Add 0.5-1 mM EDTA to chelate metal ions that could inhibit activity

• Maintain pH between 7.0-8.0 throughout purification

• Keep temperature at 4°C during all purification steps

• Consider adding protease inhibitors to prevent degradation

This protocol typically yields protein with >95% purity suitable for enzymatic and structural studies .

How does the substrate specificity of PSPTO_3837 compare with other Maf family proteins, and what structural elements determine this specificity?

The substrate specificity of PSPTO_3837 can be analyzed through comparative structural and functional studies with other Maf proteins. Based on studies of related Maf proteins, specificity is likely determined by key residues in the active site .

Comparative Substrate Specificity Analysis:

Structural elements that likely determine specificity include:

• The conserved S-R-E-K-D-K motif forms the active site core

• Variable residues surrounding this core create distinct binding pockets

• Loop regions that can adopt different conformations upon substrate binding

To experimentally determine specificity, researchers should:

• Perform site-directed mutagenesis of predicted specificity-determining residues

• Conduct comparative kinetic analyses (kcat/Km) with various substrates

• Obtain crystal structures with different bound substrates or substrate analogs

• Use molecular docking and molecular dynamics simulations to predict binding modes

What role might PSPTO_3837 play in quorum sensing regulation and virulence of P. syringae pv. tomato DC3000?

PSPTO_3837 may play a significant role in quorum sensing and virulence regulation in P. syringae pv. tomato DC3000, potentially interacting with established regulatory systems.

Potential Regulatory Interactions:

• Quorum Sensing Connection: P. syringae pv. tomato DC3000 produces N-acyl homoserine lactones (AHLs) as quorum sensing signals, regulated by PsyR (a LuxR-type regulator) and PsyI (AHL synthase) . PSPTO_3837 might modulate this signaling pathway through nucleotide metabolism or direct interaction.

• Virulence Regulation: The PsrA regulator in DC3000 controls expression of virulence factors and the hypersensitive response . If PSPTO_3837 interacts with this pathway, it could affect pathogenicity.

Experimental Approaches to Investigate This Role:

• Create PSPTO_3837 knockout mutants and assess:

  • AHL production levels

  • Expression of virulence genes (using qRT-PCR)

  • Ability to elicit the hypersensitive response in plants

  • Virulence in tomato and Arabidopsis models

• Perform transcriptome analysis comparing wild-type and ΔPSPTO_3837 strains under:

  • Different growth phases

  • Plant infection conditions

  • Oxidative stress (as Maf proteins may respond to oxidative conditions)

• Investigate protein-protein interactions between PSPTO_3837 and known virulence regulators using:

  • Bacterial two-hybrid assays

  • Co-immunoprecipitation

  • Surface plasmon resonance

The regulatory framework of P. syringae pv. tomato DC3000 includes complex interactions between PsrA, AefR, and RpoS, which collectively regulate quorum sensing and virulence . Understanding how PSPTO_3837 fits into this network would provide valuable insights into bacterial pathogenicity mechanisms.

How can structural biology techniques be applied to elucidate the catalytic mechanism of PSPTO_3837?

Advanced structural biology techniques can reveal the catalytic mechanism of PSPTO_3837 with atomic-level precision:

Comprehensive Structural Biology Workflow:

• X-ray Crystallography:

  • Crystallize PSPTO_3837 in apo form and with substrate analogs/inhibitors

  • Resolve structures at high resolution (<2.0 Å)

  • Identify catalytic residues and binding pocket architecture

  • Crystallize site-directed mutants to confirm the role of specific residues

• Cryo-Electron Microscopy:

  • Useful if the protein forms larger complexes or is difficult to crystallize

  • Can capture different conformational states during catalysis

• NMR Spectroscopy:

  • Analyze protein dynamics in solution

  • Perform chemical shift perturbation experiments to map substrate binding

  • Study conformational changes upon substrate binding

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Identify regions with altered solvent accessibility during catalysis

  • Map conformational changes upon substrate binding

• Molecular Dynamics Simulations:

  • Model substrate binding and reaction trajectory

  • Predict transition states and energy barriers

  • Simulate effects of mutations on catalytic efficiency

Based on studies of related Maf proteins, the catalytic mechanism likely involves:

• A nucleophilic attack on the α-phosphate of the nucleotide substrate

• Coordination of divalent metal ions (Mg²⁺ or Mn²⁺) to stabilize the reaction intermediate

• Specific interactions between the conserved S-R-E-K-D-K motif and the substrate

What approaches can be used to investigate the role of PSPTO_3837 in plant-pathogen interactions during infection?

Investigating PSPTO_3837's role in plant-pathogen interactions requires a multi-faceted approach combining molecular genetics, cell biology, and plant pathology techniques:

Comprehensive Investigation Strategy:

• Generation of Bacterial Mutants:

  • Create clean deletion mutants (ΔPSPTO_3837)

  • Develop complementation strains (ΔPSPTO_3837 + PSPTO_3837)

  • Engineer point mutations in critical residues

  • Create reporter fusion strains (PSPTO_3837-GFP, PSPTO_3837 promoter-luciferase)

• Plant Infection Studies:

  • Compare virulence of wild-type, mutant, and complemented strains in:

    • Tomato plants (natural host)

    • Arabidopsis thaliana (model host)

    • Nicotiana benthamiana (when lacking hopQ1-1)

  • Quantify bacterial growth in planta over time

  • Assess disease symptom development and progression

• Microscopy and Cellular Localization:

  • Use confocal microscopy with fluorescent reporter strains to track:

    • Bacterial localization during infection

    • Protein localization within bacterial cells

    • Temporal expression patterns during infection

• Host Response Analysis:

  • Compare plant defense responses to wild-type and mutant strains:

    • ROS production

    • Callose deposition

    • Defense gene expression

    • Hypersensitive response

• Interactome Studies:

  • Identify plant proteins that interact with PSPTO_3837 using:

    • Yeast two-hybrid screening

    • Co-immunoprecipitation followed by mass spectrometry

    • Bimolecular fluorescence complementation in planta

The specific type III secretion system of P. syringae pv. tomato DC3000 delivers effector proteins into plant cells to suppress host immunity . Determining whether PSPTO_3837 influences this system or directly interacts with host factors would provide valuable insights into the molecular mechanisms of bacterial pathogenesis.

How do posttranslational modifications affect the activity and stability of recombinant PSPTO_3837?

Posttranslational modifications (PTMs) can significantly impact both the activity and stability of recombinant Maf-like proteins such as PSPTO_3837. Though bacterial proteins typically have fewer PTMs than eukaryotic proteins, several modifications may be relevant:

Investigation of Potential PTMs:

• Phosphorylation:

  • Maf proteins in eukaryotes are regulated by phosphorylation on serine, threonine, and tyrosine residues

  • In bacteria, phosphorylation may occur through two-component signaling systems

  • Methods for detection:

    • Mass spectrometry (LC-MS/MS) with phosphopeptide enrichment

    • Phospho-specific antibodies (if available)

    • Phos-tag SDS-PAGE

• Oxidation of Cysteine Residues:

  • May form disulfide bonds or undergo reversible oxidation

  • Can regulate protein activity in response to oxidative stress

  • Detection methods:

    • Mass spectrometry with differential alkylation

    • Ellman's reagent for free thiol quantification

    • Diagonal electrophoresis

• Proteolytic Processing:

  • N-terminal or C-terminal processing may occur

  • Detection methods:

    • N-terminal sequencing (Edman degradation)

    • MALDI-TOF mass spectrometry

    • Western blotting with domain-specific antibodies

Experimental Approach to Study PTM Effects:

To assess stability changes resulting from PTMs, researchers should employ thermal shift assays, limited proteolysis, and long-term storage studies under various conditions. For activity assessment, standard nucleotide pyrophosphatase assays comparing modified and unmodified versions of the protein would provide quantitative data on the functional impact of specific PTMs.

What bioinformatic approaches can predict functional partners and evolutionary relationships of PSPTO_3837 within the Pseudomonas genus?

Advanced bioinformatic approaches can provide valuable insights into the functional partners and evolutionary context of PSPTO_3837:

Comprehensive Bioinformatic Analysis Strategy:

• Phylogenetic Analysis:

  • Construct maximum likelihood or Bayesian phylogenetic trees of Maf-like proteins across:

    • Different Pseudomonas species

    • Plant pathogenic bacteria

    • Model organisms with characterized Maf proteins

  • Analyze patterns of sequence conservation using tools like CONSURF

  • Identify evolutionary rates and selective pressures using dN/dS analysis

• Protein Domain Analysis:

  • Identify conserved domains and motifs using InterPro, Pfam, and PROSITE

  • Compare domain architecture with characterized Maf proteins like YhdE and YceF

  • Predict functionally important residues based on evolutionary conservation

• Genomic Context Analysis:

  • Examine gene neighborhoods across Pseudomonas genomes

  • Identify consistently co-located genes (potential functional partners)

  • Analyze operon structures and regulatory elements

• Protein-Protein Interaction Prediction:

  • Use tools like STRING, PSICQUIC, and PrePPI

  • Identify conserved interaction partners across species

  • Predict interaction interfaces using tools like PRISM or HADDOCK

• Co-expression Analysis:

  • Mine transcriptomic datasets to identify genes with similar expression patterns

  • Focus on plant infection conditions and stress responses

  • Construct co-expression networks to identify functional modules

Example of Phylogenetic Classification:

The functional prediction would be enhanced by integrating these computational approaches with experimental data from genetic screens, such as the competitive selection methodology used to identify novel flagellar motility genes in P. syringae pv. tomato DC3000 .