Recombinant Pseudomonas syringae pv. tomato Cytidylate kinase (cmk)

What is the genomic context of the cytidylate kinase gene in Pseudomonas syringae pv. tomato DC3000?

Cytidylate kinase in P. syringae pv. tomato DC3000 is encoded within the bacterial chromosome. The gene is part of the nucleotide biosynthesis pathway and is typically found in a genomic neighborhood with other genes involved in nucleotide metabolism. Unlike effector proteins that are regulated by the HrpL alternative sigma factor, cmk is constitutively expressed as part of the core metabolic functions . The gene is essential for bacterial viability since it catalyzes a critical step in the pyrimidine biosynthesis pathway, making it an interesting target for understanding bacterial metabolism during infection processes.

How does the structure of P. syringae pv. tomato cmk compare to other bacterial cytidylate kinases?

While the specific crystal structure of P. syringae pv. tomato cmk has not been fully characterized in the provided search results, structural analysis approaches similar to those used for the GntR family transcriptional regulator from P. syringae pv. tomato DC3000 could be applied . Bacterial cytidylate kinases typically contain a nucleotide-binding domain with conserved motifs for ATP binding and catalysis. Comparative structural analysis would likely reveal high conservation in the active site region while potentially showing species-specific variations in peripheral domains. These structural differences could be exploited for the development of specific inhibitors targeting P. syringae metabolism.

What expression systems are most effective for producing recombinant P. syringae pv. tomato cmk?

For effective production of recombinant P. syringae pv. tomato cmk, E. coli-based expression systems are generally recommended. The pET expression system under the control of T7 promoter has proven successful for many bacterial enzymes. Key considerations include:

• Codon optimization for E. coli (or alternative host)

• Addition of affinity tags (His6-tag is commonly used) for purification

• Temperature optimization (often 16-18°C) to enhance protein solubility

• Use of bacterial strains with extra copies of rare tRNAs (like Rosetta or CodonPlus)

Expression can be verified using SDS-PAGE analysis followed by Western blotting with anti-His antibodies if a His-tag is incorporated. For purification, immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography typically yields highly pure protein suitable for enzymatic and structural studies .

How can cmk be utilized to study metabolic changes during P. syringae infection of plant hosts?

Cytidylate kinase serves as an ideal metabolic marker for studying changes in nucleotide metabolism during plant infection. Researchers can use recombinant cmk to:

• Develop activity assays that measure nucleotide flux during different infection stages

• Create fluorescently-tagged cmk variants to visualize metabolic compartmentalization during infection

• Perform metabolomic studies comparing wild-type and cmk-modified strains

For experimental design, researchers could isolate bacteria from infected plant tissues at different timepoints (early infection, established infection, systemic spread) and measure cmk activity in correlation with bacterial load and symptom development. This approach could reveal how nucleotide metabolism adapts during different infection phases and in response to host defenses .

What is the potential interplay between cmk activity and the type III secretion system in P. syringae pv. tomato DC3000?

While cmk is primarily a metabolic enzyme, its activity may indirectly influence virulence mechanisms like the type III secretion system (T3SS). The T3SS in P. syringae pv. tomato DC3000 requires substantial energy for assembly and operation, which is derived from nucleotide metabolism . Experimental approaches to investigate this connection could include:

• Creating conditional cmk mutants to observe effects on effector protein production and secretion

• Measuring ATP/GTP pools in wild-type versus cmk-modified strains during T3SS activation

• Analyzing transcriptional coupling between metabolic and virulence genes during infection

This theoretical data table illustrates potential relationships between cmk activity and T3SS function that could be experimentally verified .

How might cmk be involved in bacterial response to plant immunity triggers?

P. syringae pv. tomato must adapt its metabolism when facing plant immune responses. When plants detect bacterial pathogens through pattern recognition receptors or effector-triggered immunity, they often produce antimicrobial compounds and reactive oxygen species that can damage bacterial DNA and disrupt nucleotide pools.

Experimental approaches to study cmk's role in this adaptation could include:

• Exposing P. syringae to plant immune elicitors and measuring changes in cmk expression and activity

• Comparing survival rates of wild-type versus cmk-modified strains when exposed to plant defense molecules

• Analyzing whether cmk activity correlates with bacterial persistence in resistant versus susceptible plant varieties

Research could examine whether cmk upregulation helps bacteria repair damage to nucleotide pools caused by plant immune responses, potentially connecting basic metabolism to pathogen survival strategies.

What are the optimal conditions for measuring recombinant P. syringae pv. tomato cmk enzymatic activity?

For reliable measurement of recombinant P. syringae pv. tomato cmk activity, a coupled spectrophotometric assay is recommended. The standard reaction mixture should contain:

• 50 mM Tris-HCl (pH 7.5)

• 50 mM KCl

• 5 mM MgCl₂

• 1 mM ATP

• 0.5 mM CMP

• 0.2 mM NADH

• Coupling enzymes (pyruvate kinase and lactate dehydrogenase)

• 0.5-5 μg purified recombinant cmk

The reaction is monitored by the decrease in NADH absorbance at 340 nm, which correlates with ADP production as cmk phosphorylates CMP to CDP. Key parameters to optimize include:

• pH range (typically 7.2-8.0)

• Magnesium concentration (critical for ATP binding)

• Temperature (usually 25-37°C)

• Substrate concentrations for kinetic analysis

Enzyme kinetics should be determined under steady-state conditions with varying concentrations of both ATP and CMP to generate Lineweaver-Burk plots for determining Km and Vmax values .

How can protein-protein interactions between cmk and other bacterial proteins be studied?

To investigate potential protein-protein interactions involving cmk in P. syringae pv. tomato, multiple complementary approaches should be employed:

• Bacterial Two-Hybrid System: This approach can be used to screen for potential interaction partners in vivo. By fusing cmk to one domain of a split reporter protein and creating a library of bacterial proteins fused to the complementary domain, interactions can be detected through reporter activation.

• Co-Immunoprecipitation: Using antibodies against either native cmk or epitope tags on recombinant cmk, protein complexes can be isolated from bacterial lysates and analyzed by mass spectrometry to identify interaction partners.

• Biolayer Interferometry or Surface Plasmon Resonance: These techniques can measure direct binding between purified recombinant cmk and candidate interacting proteins, providing quantitative binding constants.

• Crosslinking Mass Spectrometry: This advanced approach can capture transient interactions and precisely map interaction interfaces through chemical crosslinking followed by mass spectrometry analysis.

When designing these experiments, it's important to consider that cmk may interact with components of metabolic complexes, nucleotide synthesis enzymes, or potentially regulatory proteins that coordinate metabolism with virulence .

What approaches can be used to study the impact of cmk inhibition on P. syringae virulence?

Investigating the relationship between cmk inhibition and P. syringae virulence requires multi-faceted approaches:

• Generation of Conditional Mutants: Since cmk is likely essential, temperature-sensitive mutants or inducible knockdown strains should be created rather than complete knockouts.

• Chemical Inhibition Studies: Identify specific inhibitors of bacterial cmk that have minimal effect on plant nucleotide metabolism. Test these in planta during infection processes.

• Plant Infection Assays: Compare bacterial growth curves between wild-type and cmk-inhibited strains using methods similar to those employed in the YDA kinase studies . Key measurements include:

  • Bacterial population dynamics (CFU/cm²)

  • Disease symptom development

  • Expression of virulence genes

  • Effector protein translocation efficiency

• Metabolomic Analysis: Compare nucleotide pools between wild-type and cmk-inhibited strains during infection to correlate metabolic changes with virulence.

This hypothetical data illustrates how cmk inhibition might correlate with reduced virulence parameters, which could be experimentally verified using approaches similar to those used in studying YDA kinase effects on plant immunity .

How can the substrate specificity of P. syringae pv. tomato cmk be determined?

Determining substrate specificity of recombinant P. syringae pv. tomato cmk involves systematic biochemical characterization:

• Substrate Range Testing: Assess activity with various nucleoside monophosphates (CMP, UMP, AMP, GMP, IMP) using standardized assay conditions to determine substrate preference.

• Kinetic Parameter Determination: Calculate Km, kcat, and catalytic efficiency (kcat/Km) for each viable substrate to quantitatively rank substrate preferences.

• pH and Temperature Profiling: Determine optimal conditions and how they might differ for different substrates.

• Mutational Analysis: Create site-directed mutants of key active site residues to map the structural determinants of substrate specificity.

• Computational Modeling: If structural data becomes available, molecular docking and molecular dynamics simulations can predict substrate binding modes and energetics.

Results should be presented as comprehensive kinetic parameters:

This hypothetical data table illustrates how substrate specificity would be characterized and reported in a comprehensive enzymatic study .

What structural features differentiate P. syringae pv. tomato cmk from plant cytidylate kinases?

Understanding structural differences between bacterial and plant cytidylate kinases is crucial for developing pathogen-specific inhibitors. Comparative structural analysis should focus on:

• Primary Sequence Analysis: Alignment of P. syringae cmk with plant homologs to identify conserved catalytic residues versus divergent regions.

• 3D Structural Comparison: If crystal structures are available (similar to the approach used for the GntR family transcriptional regulator ), superimpose bacterial and plant enzymes to identify unique pockets or conformational differences.

• Active Site Architecture: Compare substrate binding sites, identifying amino acid differences that could be exploited for selective inhibition.

• Surface Electrostatics: Analyze the electrostatic potential distributions that might affect inhibitor binding differently between bacterial and plant enzymes.

• Allosteric Sites: Identify potential regulatory sites present in bacterial cmk that may be absent in plant homologs.

Key structural differences, once identified, could be presented in a table highlighting potential target sites for selective inhibitors:

How could cmk inhibitors be used in conjunction with plant immunity enhancers?

Cytidylate kinase inhibitors could potentially synergize with plant immunity enhancers in a dual-action approach to control P. syringae infections. This strategy would combine metabolic disruption of the pathogen with strengthened host defenses:

• Mechanism of Synergy: While cmk inhibitors would disrupt bacterial nucleotide metabolism, plant immunity enhancers (like those targeting the YDA kinase pathway ) would simultaneously boost plant defense responses, creating a multi-pronged attack.

• Experimental Design: Tests should include:

  • Sequential treatments (immunity enhancer followed by cmk inhibitor)

  • Simultaneous treatments

  • Concentration optimization for both components

  • Assessment of bacterial growth suppression and plant health parameters

• Expected Outcomes: The combination therapy could potentially:

  • Reduce effective concentrations of both components

  • Decrease likelihood of resistance development

  • Provide more durable protection

One approach would be to combine cmk inhibitors with enhancers of the YDA kinase pathway, which has been shown to control immune responses in both Arabidopsis and tomato against P. syringae pv. tomato DC3000 .

What are the considerations for developing recombinant cmk as a tool for understanding bacterial metabolism during infection?

Developing recombinant cmk as a research tool requires addressing several key considerations:

• Expression and Purification Optimization:

  • Design constructs with various affinity tags (His, GST, MBP)

  • Optimize soluble expression conditions

  • Develop multi-step purification protocols for highest purity

  • Ensure stability during storage (buffer conditions, additives)

• Functional Characterization:

  • Develop robust activity assays adaptable to different experimental conditions

  • Validate enzyme parameters match native cmk behavior

  • Create inactive mutants as controls

• Tool Development Applications:

  • Engineer substrate-specific variants for metabolic pathway tracing

  • Develop cmk-based biosensors to monitor nucleotide pools in vivo

  • Create fluorescently-labeled variants for localization studies

• In Planta Applications:

  • Design delivery methods for recombinant cmk into plant tissues

  • Develop assays to measure cmk activity in planta during infection

  • Create protocols for extracting bacterial proteins from plant tissue without losing activity

This approach would leverage expertise in protein biochemistry to create versatile tools for studying P. syringae metabolism similar to how the calmodulin-dependent adenylate cyclase (Cya) reporter system was optimized for studying the translocation of P. syringae TTSS effectors .