Recombinant Pseudomonas syringae pv. tomato Ribose-phosphate pyrophosphokinase (prs)

What is Ribose-phosphate pyrophosphokinase (prs) in Pseudomonas syringae pv. tomato?

Ribose-phosphate pyrophosphokinase (prs) in Pseudomonas syringae pv. tomato is an essential enzyme that catalyzes the conversion of ribose-5-phosphate (R5P) to 5-phosphoribosyl-1-pyrophosphate (PRPP). The enzyme plays a crucial role in nucleotide biosynthesis pathways and is identified in proteomics studies of P. syringae pv. tomato strain DC3000 (ATCC BAA-871) . The enzyme is encoded by the prs gene and is fundamental to bacterial metabolism, particularly in pathways related to nucleotide synthesis, histidine biosynthesis, and tryptophan biosynthesis. In P. syringae, this enzyme connects the pentose phosphate pathway (PPP) with nucleotide biosynthesis by facilitating the conversion of R5P, a product of PPP, into PRPP, which serves as a precursor for nucleotide synthesis.

What is the functional significance of prs in P. syringae metabolism?

The prs enzyme occupies a critical metabolic junction in P. syringae, connecting carbohydrate metabolism with nucleic acid synthesis. It catalyzes the first committed step in the de novo and salvage pathways of purine and pyrimidine nucleotide synthesis by producing PRPP . This reaction requires ATP as a phosphate donor, making it energy-dependent. The reaction can be represented as:

Ribose-5-phosphate + ATP → 5-phosphoribosyl-1-pyrophosphate + AMP

The enzyme's activity is particularly important during bacterial growth and infection cycles when rapid DNA synthesis is required. The regulation of prs activity affects the availability of nucleotides for DNA replication, RNA synthesis, and other essential cellular processes. In pathogenic contexts, prs activity may be upregulated to support the increased metabolic demands during infection and proliferation within host tissues .

How does the structure of P. syringae prs compare to homologous enzymes in other bacteria?

P. syringae pv. tomato prs shares significant structural similarities with homologous enzymes from other bacterial species while maintaining some unique features. The enzyme contains conserved domains characteristic of the ribose-phosphate pyrophosphokinase family, including nucleotide-binding sites and catalytic residues. Based on proteomics data, the P. syringae prs has been identified in various experimental conditions, with varying expression levels depending on growth conditions and environmental stressors .

The active enzyme typically functions as an oligomer, with activity often dependent on proper quaternary structure formation. While specific crystallographic data for P. syringae prs is limited, comparative analysis with characterized homologs suggests the presence of allosteric regulation sites that respond to nucleotide levels, similar to the feedback inhibition observed in human PRPS enzymes by purine nucleotides .

What are optimal methods for recombinant expression and purification of P. syringae prs?

For recombinant expression of P. syringae pv. tomato prs, researchers typically clone the prs gene into expression vectors such as pET systems with appropriate tags (His-tag or FLAG-tag) for purification purposes . The following methodological approach is recommended:

• Gene Amplification and Cloning:

  • PCR-amplify the prs gene from P. syringae pv. tomato genomic DNA using high-fidelity polymerase

  • Clone into an expression vector between appropriate restriction sites (e.g., EcoRI and HindIII)

  • Verify the sequence to ensure no mutations were introduced

• Expression Conditions:

  • Transform into an E. coli expression strain (BL21(DE3) or similar)

  • Culture in LB medium at 37°C until OD600 reaches 0.6-0.8

  • Induce with IPTG (0.5-1 mM) at lower temperature (16-25°C) for 4-16 hours to enhance soluble protein production

• Purification Strategy:

  • Harvest cells and lyse using sonication or French press in appropriate buffer (e.g., 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

  • Purify using affinity chromatography (Ni-NTA for His-tagged protein)

  • Further purify using size exclusion chromatography to obtain homogeneous protein preparation

  • Assess purity by SDS-PAGE and enzyme activity tests

The purified enzyme should be stored in buffer containing stabilizing agents like glycerol at -80°C to maintain activity.

How can researchers accurately measure P. syringae prs enzymatic activity?

The enzymatic activity of P. syringae prs can be measured using several complementary approaches:

• Direct Product Formation Assay:

  • Measure the formation of PRPP using chromatographic methods (HPLC)

  • Reaction mixture typically contains:

    • Ribose-5-phosphate (1-2 mM)

    • ATP (2-5 mM)

    • MgCl₂ (5-10 mM)

    • Buffer (50 mM Tris-HCl, pH 7.5-8.0)

    • Purified enzyme (0.1-1 μg)

• Coupled Enzyme Assay:

  • Couple PRPP formation to subsequent enzymatic reactions that can be measured spectrophotometrically

  • Example: PRPP + orotate → orotidine-5'-monophosphate (OMP) + PPi, catalyzed by orotate phosphoribosyltransferase

  • OMP formation can be monitored at 295 nm

• Radiometric Assay:

  • Use ¹⁴C-labeled ribose-5-phosphate as substrate

  • Separate the ¹⁴C-PRPP product by thin-layer chromatography or ion-exchange chromatography

  • Quantify radioactivity using scintillation counting

For all assays, proper controls should be included, such as enzyme-free reactions and heat-inactivated enzyme preparations. Kinetic parameters (Km, Vmax) can be determined by varying substrate concentrations and analyzing the data using Michaelis-Menten or Lineweaver-Burk plots.

What approaches are effective for studying prs regulation in P. syringae?

Studying the regulation of prs in P. syringae requires a multi-faceted approach combining genetic, biochemical, and systems biology methods:

• Transcriptional Regulation:

  • RT-qPCR to measure prs mRNA levels under different conditions, similar to the approaches used for studying GABA metabolic genes in PsPto

  • Promoter-reporter fusions (e.g., prs promoter-GFP) to monitor expression in vivo

  • ChIP-seq to identify transcription factors binding to the prs promoter region

• Post-translational Regulation:

  • Mass spectrometry to identify potential phosphorylation, acetylation, or other modifications

  • Western blotting with phospho-specific antibodies if phosphorylation is suspected

  • Activity assays in the presence of potential allosteric regulators (nucleotides, metabolic intermediates)

• Metabolic Regulation:

  • Metabolomics to correlate prs activity with metabolite concentrations

  • Isotope tracing experiments to track carbon flow through the pathway

  • Mathematical modeling to integrate enzyme kinetics with cellular metabolism

• Environmental Response:

  • Monitor prs expression and activity during plant infection processes

  • Compare responses in different growth media and stress conditions

  • Analyze correlation with bacterial virulence factors

For example, researchers could adapt methods similar to those used to study GABA metabolism in P. syringae, where gene expression was analyzed after exposure to plant-derived compounds in concentrations matching those found in plant tissues .

How does prs activity contribute to P. syringae virulence in tomato plants?

The contribution of prs to P. syringae virulence likely involves multiple interconnected mechanisms:

• Support for Rapid Proliferation:

  • Prs provides PRPP for nucleotide synthesis, supporting the rapid bacterial proliferation required during infection

  • The enzyme's activity may be upregulated during infection to meet increased demands for DNA and RNA synthesis

• Connection to Signaling Pathways:

  • Nucleotides produced through prs-dependent pathways may serve as signaling molecules or precursors for secondary metabolites involved in virulence

  • These pathways could interact with chemosensory systems like the PsPto-PscC chemoreceptor system that detects plant-derived compounds such as GABA and L-Pro

• Metabolic Adaptation:

  • Prs activity may enable metabolic flexibility, allowing P. syringae to adapt to the unique nutritional environment of the plant apoplast

  • The enzyme could help balance carbon flux between energy production and biosynthetic pathways during different infection stages

• Potential Interaction with Plant Defense:

  • Nucleotide metabolism in bacteria may interact with plant defense signaling, potentially through mechanisms similar to how GABA and L-Pro levels increase during infection and participate in plant defense regulation

Research suggests that P. syringae must efficiently coordinate chemotaxis, entry mechanisms, and metabolism to successfully establish infection. While direct evidence linking prs to these processes is limited, its central role in nucleotide metabolism suggests it would be critical for supporting the metabolic demands of infection processes.

What is the relationship between prs activity and nucleotide pools during different growth phases?

The relationship between prs activity and nucleotide pools in P. syringae likely follows patterns similar to those observed in other organisms, but with pathogen-specific adaptations:

• Growth Phase-Dependent Regulation:

  • Prs activity is typically higher during exponential growth phases when DNA synthesis is most active

  • This pattern aligns with observations that nucleotide concentrations increase from late G1 to S phase and decrease after completion of DNA duplication in eukaryotic systems

  • In P. syringae, this would correspond to periods of rapid proliferation after successful host entry

• Feedback Regulation:

  • Similar to human PRPS enzymes, bacterial prs is likely subject to feedback inhibition by purine nucleotides (AMP, GMP)

  • This regulation prevents overproduction of nucleotides and maintains balanced pools

  • The specific regulatory mechanisms in P. syringae may be adapted to its unique metabolic needs during plant infection

• Metabolic Integration:

  • Prs connects the pentose phosphate pathway to nucleotide synthesis, allowing coordination between carbon metabolism and nucleic acid production

  • During infection, this coordination may be critical for balancing energy production with the biosynthetic demands of rapid growth

How does the pentose phosphate pathway interact with nucleotide synthesis via prs during infection?

The interaction between the pentose phosphate pathway (PPP) and nucleotide synthesis through prs represents a critical metabolic junction during P. syringae infection:

• Carbon Flux Coordination:

  • During infection, P. syringae must balance carbon utilization between energy production (glycolysis, TCA cycle) and biosynthetic pathways

  • The oxidative branch of PPP generates NADPH for biosynthetic reactions and defense against oxidative stress

  • The non-oxidative branch produces ribose-5-phosphate, which prs converts to PRPP for nucleotide synthesis

  • This coordination ensures sufficient nucleotide production while maintaining energy generation

• Adaptation to Plant Environment:

  • The plant apoplast provides a specific nutritional environment that may influence PPP activity

  • P. syringae may modulate PPP and prs activities in response to available carbon sources and plant defense responses

  • This modulation could involve shifts between the oxidative and non-oxidative branches of PPP to optimize ribose-5-phosphate production

• Response to Oxidative Stress:

  • During plant defense responses, P. syringae encounters reactive oxygen species

  • Increased flux through the oxidative PPP generates NADPH for antioxidant systems

  • This shift may alter ribose-5-phosphate availability for prs, potentially creating a regulatory mechanism linking oxidative stress defense to nucleotide synthesis

The specific mechanisms coordinating these pathways during infection remain areas for further research, but understanding them could reveal potential targets for disease management strategies.

How should researchers interpret proteomics data for P. syringae prs expression?

Interpreting proteomics data for P. syringae prs requires careful consideration of several factors:

• Quantitative Analysis:

  • Absolute quantification methods (like those in the proteomics data) provide valuable information about protein abundance

  • When analyzing prs expression data, consider:

    • Raw intensity values (e.g., 504,220,000 for NADH-quinone oxidoreductase subunit I)

    • Normalized abundances across conditions

    • Statistical significance of observed differences

• Comparative Analysis:

  • Compare prs abundance across different:

    • Growth conditions (minimal vs. rich media)

    • Infection stages (early vs. established infection)

    • Bacterial strains (wild-type vs. mutants)

  • Look for co-regulated proteins that may indicate functional relationships

• Integration with Other Data Types:

  • Correlate protein abundance with:

    • Transcriptomic data (mRNA levels)

    • Metabolomics data (substrate/product concentrations)

    • Phenotypic observations (growth rates, virulence)

• Biological Context:

  • Consider how prs expression fits within larger metabolic networks

  • Examine expression patterns of enzymes in connected pathways

  • Interpret changes in the context of bacterial adaptation to specific environments

Table 1: Example interpretation framework for P. syringae prs proteomics data

When analyzing proteomics data like that presented in search result , researchers should focus on both the absolute values and the relative changes across experimental conditions, while considering the biological context of nucleotide metabolism in pathogenesis.

What statistical approaches are recommended for analyzing prs expression data?

When analyzing prs expression data from experiments involving P. syringae, researchers should employ robust statistical methods appropriate for the specific data type and experimental design:

• For Transcriptomic Data (qRT-PCR, RNA-Seq):

  • Normalization using appropriate reference genes (similar to the approach used for GABA metabolic genes)

  • Fold change calculations with error propagation

  • Statistical tests:

    • Student's t-test for simple two-condition comparisons

    • ANOVA with post-hoc tests (Tukey's HSD) for multi-condition experiments

    • Consider non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) if data is not normally distributed

• For Proteomics Data:

  • Log transformation of intensity values to achieve normal distribution

  • Appropriate normalization to account for technical variation

  • Statistical methods:

    • Differential expression analysis using limma or similar tools

    • Multiple testing correction (Benjamini-Hochberg FDR)

    • Clustering approaches to identify co-regulated proteins

• For Enzymatic Activity Data:

  • Michaelis-Menten or Lineweaver-Burk analysis for kinetic parameters

  • ANOVA for comparing activity across conditions

  • Regression analysis for identifying correlations with metabolite concentrations

• For Multi-omics Integration:

  • Correlation analysis between transcriptomic, proteomic, and metabolomic data

  • Principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA)

  • Network analysis to identify functional relationships

When reporting results, include both the statistical significance (p-values with appropriate corrections) and the biological significance (fold changes, effect sizes) to provide a complete picture of the observed effects.

How can researchers reconcile contradictory findings about prs function across different experimental systems?

Researchers frequently encounter seemingly contradictory findings about enzyme function when working across different experimental systems. For P. syringae prs, consider these strategies for reconciliation:

• Methodological Differences:

  • Carefully examine experimental conditions:

    • Buffer composition, pH, temperature, and ionic strength

    • Substrate concentrations and purity

    • Enzyme preparation methods (tags, fusion partners, purification procedures)

  • Standardize protocols when possible or explicitly account for methodological variations

• Biological Context Variations:

  • Growth conditions affect bacterial physiology:

    • Nutrient availability alters metabolic flux

    • Growth phase impacts enzyme expression and regulation

    • Plant-derived compounds may influence bacterial metabolism

  • Use defined media and consistent growth parameters to reduce variability

• Strain Differences:

  • Genetic variations between P. syringae strains can affect prs function:

    • Compare sequences to identify polymorphisms

    • Examine genomic context for differences in regulatory elements

    • Consider horizontal gene transfer events that might affect enzyme function

  • Use reference strains (e.g., DC3000) and document strain provenance

• Multi-scale Approach:

  • Integrate data from different experimental scales:

    • In vitro biochemical assays provide mechanistic insights

    • Cellular studies capture physiological context

    • Plant infection models reveal in vivo relevance

    • Computational modeling can help reconcile disparate observations

• Statistical Analysis:

  • Meta-analysis of multiple studies

  • Bayesian approaches to integrate prior knowledge with new data

  • Sensitivity analysis to identify key parameters driving different outcomes

When documenting research findings, explicitly discuss potential sources of variation and place results in the context of existing literature, acknowledging both agreements and discrepancies.

What are promising approaches for studying the role of prs in P. syringae adaptation to plant environments?

Several innovative approaches show promise for elucidating the role of prs in P. syringae adaptation to plant environments:

• Spatiotemporal Expression Analysis:

  • Use fluorescent reporter fusions to visualize prs expression during plant infection

  • Apply single-cell technologies to examine heterogeneity in bacterial populations

  • Develop biosensors for real-time monitoring of nucleotide pools in bacterial cells

  • These approaches would provide insights into how prs activity changes throughout the infection process

• Systems Biology Integration:

  • Develop genome-scale metabolic models specific to P. syringae during plant infection

  • Integrate transcriptomic, proteomic, and metabolomic data in a unified framework

  • Apply flux balance analysis to predict metabolic adaptations during infection

  • These methods could reveal how prs activity is coordinated with broader metabolic networks

• Chemical Biology Approaches:

  • Develop specific inhibitors of P. syringae prs

  • Use activity-based protein profiling to monitor prs activity in vivo

  • Apply metabolic labeling strategies to track nucleotide synthesis during infection

  • These tools would allow precise manipulation and monitoring of prs function

• Comparative Studies:

  • Analyze prs function across different pathovars of P. syringae with varying host ranges

  • Compare prs activity in bacterial strains with different virulence levels

  • Examine plant cultivars with varying susceptibility to P. syringae infection

  • Such comparisons could reveal host-specific adaptations in nucleotide metabolism

These approaches, particularly when combined, have the potential to significantly advance our understanding of how P. syringae adapts its nucleotide metabolism during plant infection, potentially revealing new targets for disease management strategies.

How might CRISPR-Cas9 genome editing be applied to study prs function in P. syringae?

CRISPR-Cas9 technology offers powerful approaches for investigating prs function in P. syringae through precise genetic manipulation:

• Gene Knockout and Complementation:

  • Generate complete prs deletion mutants to assess essentiality

  • Create conditional knockouts using inducible promoters if prs is essential

  • Complement mutants with wild-type or modified prs variants

  • These manipulations would reveal the phenotypic consequences of prs deficiency

• Point Mutations and Domain Analysis:

  • Introduce specific point mutations in catalytic residues

  • Create domain deletions or swaps to examine functional regions

  • Generate allelic series with varying enzyme activity levels

  • These precise modifications would provide insights into structure-function relationships

• Regulatory Element Editing:

  • Modify prs promoter regions to alter expression patterns

  • Disrupt potential transcription factor binding sites

  • Engineer inducible or constitutive expression systems

  • These approaches would clarify transcriptional regulation mechanisms

• Protein Tagging for In Vivo Studies:

  • Insert epitope tags or fluorescent protein fusions at the genomic locus

  • Create split protein complementation systems to study protein-protein interactions

  • Develop degron tags for controlled protein degradation

  • These tools would enable monitoring of prs localization, interactions, and turnover

When applying CRISPR-Cas9 in P. syringae, researchers should optimize protocols for this specific bacterium, considering factors such as transformation efficiency, homologous recombination capability, and potential off-target effects. The technology could be particularly valuable for creating subtle modifications that would be difficult to achieve using traditional genetic approaches.

How does understanding P. syringae prs contribute to broader plant-microbe interaction knowledge?

Research on P. syringae prs provides insights that extend beyond this specific enzyme to enhance our understanding of plant-microbe interactions more broadly:

• Metabolic Adaptation During Infection:

  • P. syringae prs represents a model for studying how bacterial metabolism adapts to plant environments

  • Understanding how nucleotide synthesis is regulated during infection may reveal common principles applicable to other plant pathogens

  • The coordination between carbon metabolism and nucleotide synthesis likely represents a conserved challenge for plant-associated microorganisms

• Integration with Signaling Systems:

  • Similar to how the PsPto-PscC chemoreceptor system responds to plant-derived compounds like GABA and L-Pro , metabolic enzymes like prs may be integrated with sensing and signaling networks

  • This integration could represent a fundamental aspect of how bacteria perceive and respond to plant environments

• Evolution of Metabolic Capabilities:

  • Comparing prs across different P. syringae pathovars and other plant-associated bacteria could reveal how metabolic capabilities evolve during adaptation to different plant hosts

  • Such comparative analyses might identify signatures of selection that indicate key adaptations

• Targets for Intervention:

  • Central metabolic nodes like prs represent potential targets for disease management strategies

  • Understanding the essentiality and regulation of such enzymes could guide the development of targeted approaches to disrupt bacterial infection processes

By studying specific enzymes like prs within their broader biological context, researchers gain insights into the fundamental principles governing plant-microbe interactions, potentially leading to innovative approaches for promoting beneficial interactions while preventing pathogenic ones.

What are the implications of P. syringae prs research for developing novel plant disease management strategies?

Research on P. syringae prs has several potential implications for developing novel plant disease management strategies:

• Enzyme Inhibitors as Antimicrobials:

  • If prs is essential for P. syringae virulence, specific inhibitors could serve as targeted antimicrobials

  • Structure-based drug design approaches could identify compounds that selectively inhibit bacterial prs while sparing plant homologs

  • Such inhibitors might disrupt bacterial proliferation during critical infection stages

• Metabolic Priming of Plant Defense:

  • Understanding how bacterial nucleotide metabolism interacts with plant defense could reveal approaches for priming plants

  • Manipulation of plant metabolites that influence bacterial prs activity might alter infection outcomes

  • This approach could leverage natural defense mechanisms without introducing exogenous antimicrobials

• Diagnostic Tools:

  • Knowledge of prs expression patterns could inform the development of diagnostic tools

  • Molecular markers based on prs or related genes might help identify P. syringae infections earlier

  • These diagnostics could enable more timely and targeted intervention strategies

• Resistant Crop Development:

  • Insights into how P. syringae prs contributes to virulence could guide breeding or engineering approaches

  • Plants might be developed to express molecules that interfere with bacterial nucleotide metabolism

  • Alternatively, plants could be selected or engineered to alter apoplast composition in ways that disadvantage bacterial metabolism

These potential applications highlight the importance of fundamental research on bacterial metabolism for developing practical disease management strategies, illustrating how molecular understanding can translate into agricultural innovation.