Recombinant Pseudomonas syringae pv. tomato Enolase 1 (eno1)

What is the function of Enolase 1 in Pseudomonas syringae pv. tomato?

Experimental approaches to investigate eno1 function include:

• Gene knockout studies to assess growth and virulence phenotypes

• Protein localization studies using fluorescently tagged eno1

• Transcriptomics to examine expression patterns during plant infection

• Interactome mapping to identify potential protein-protein interactions

How conserved is Enolase 1 among different strains of Pseudomonas syringae?

Enolase 1 is highly conserved among Pseudomonas syringae pathovars, reflecting its essential metabolic function. Comparative genomic analyses of P. syringae strains indicate that core metabolic genes like eno1 show significantly less diversification than virulence-associated genes such as type III secreted effectors (T3SEs) . The P. syringae species complex exhibits substantial diversity with T3SEs showing evidence of horizontal gene transfer, gene gain/loss events, and positive selection . In contrast, metabolic enzymes like enolase typically display much higher sequence conservation.

A genomic study of 494 P. syringae strains isolated from over 100 hosts identified extensive variation in virulence factors but higher conservation of metabolic genes . This conservation pattern is consistent with the dual role of enolase as both a metabolic enzyme and potential virulence factor.

What analytical techniques are most effective for characterizing recombinant P. syringae pv. tomato eno1?

For comprehensive characterization of recombinant P. syringae pv. tomato eno1, a multi-method approach is most effective:

• Structural characterization:

  • SDS-PAGE for purity assessment (>85% purity is typically achieved)

  • Mass spectrometry for precise molecular weight determination and peptide mapping

  • Circular dichroism spectroscopy to evaluate secondary structure integrity

• Functional characterization:

  • Enzyme activity assay measuring the conversion of 2-phosphoglycerate to phosphoenolpyruvate

  • Temperature and pH profiling to determine optimal reaction conditions

  • Kinetic parameter determination (Km, Vmax, kcat)

• Immunological characterization:

  • Western blotting with anti-His tag antibodies for tagged proteins

  • Development of specific antibodies against P. syringae eno1 for immunodetection

  • ELISA-based quantification

For functional studies, it's crucial to validate that the recombinant protein retains native enzymatic activity. Stability assessments under different buffer conditions and storage temperatures are also essential for experimental planning .

How might recombinant eno1 be used to study P. syringae pathogenicity mechanisms?

Recombinant P. syringae pv. tomato eno1 offers several sophisticated approaches to investigate pathogenicity mechanisms:

• Host-pathogen interaction studies:

  • Pull-down assays using recombinant eno1 to identify potential plant protein binding partners

  • Surface plasmon resonance to characterize binding kinetics with plant targets

  • Yeast two-hybrid screening to discover novel protein-protein interactions

• Immunological investigations:

  • Generation of eno1-specific antibodies for in planta localization studies

  • Immunoprecipitation to isolate eno1-containing protein complexes during infection

  • Investigating whether eno1 elicits plant immune responses

• Structural biology applications:

  • Crystallography studies to determine high-resolution protein structure

  • Structure-guided mutagenesis to identify functionally important residues

  • Computational modeling of interactions with plant proteins

• Systems biology integration:

  • Incorporation of eno1 function into metabolic models of P. syringae during infection

  • Network analysis to position eno1 in pathogenicity pathways

Recent studies on type III secretion systems in P. syringae have demonstrated how individual proteins contribute to virulence . Similar methodologies could be applied to investigate potential non-canonical roles of eno1 in pathogenicity beyond its metabolic function.

How does eno1 compare to other potential virulence factors in P. syringae pv. tomato?

While type III secreted effectors (T3SEs) are the primary virulence factors in P. syringae pv. tomato , metabolic enzymes like eno1 represent a distinct class of potential virulence contributors:

Unlike specialized virulence factors such as AvrE1 and HopM1 that directly manipulate host cell processes , eno1's contribution to virulence would likely be subtler and multifaceted. Studies in other bacterial pathogens have shown that enolases can contribute to adherence, biofilm formation, and host immune evasion.

P. syringae pv. tomato employs a diverse array of virulence strategies. Race 1 strains have evolved to overcome host resistance by modifying effectors like AvrPto and AvrPtoB . Understanding how conserved metabolic enzymes like eno1 potentially contribute to virulence could reveal new aspects of the pathogen's adaptive strategy.

What experimental approaches could identify potential moonlighting functions of P. syringae pv. tomato eno1?

To identify non-glycolytic functions of P. syringae pv. tomato eno1, researchers should consider these methodological approaches:

• Subcellular localization studies:

  • Immunofluorescence microscopy with anti-eno1 antibodies to detect unexpected localization patterns

  • Cell fractionation followed by Western blotting to identify eno1 in membrane or extracellular fractions

  • GFP-fusion proteins to track localization dynamics during infection

• Protein-protein interaction mapping:

  • Co-immunoprecipitation with recombinant eno1 using plant extracts

  • Bacterial two-hybrid screening against P. syringae or plant proteome libraries

  • Cross-linking mass spectrometry to identify interaction partners in planta

• Functional genomics approaches:

  • Site-directed mutagenesis to create variants with intact catalytic activity but disrupted surface features

  • Complementation of eno1 knockout with catalytically inactive mutants to isolate non-metabolic functions

  • Conditional expression systems to separate growth phenotypes from virulence phenotypes

• Infection model studies:

  • Gene knockdowns versus catalytic mutants in plant infection assays

  • Competitive infection assays with eno1 variants

  • Transcriptome analysis of plant responses to purified recombinant eno1

Similar experimental frameworks have been successfully employed to study the non-canonical functions of other bacterial proteins, including the discovery that the P. syringae effector HopM1 has unexpected functions in suppressing plant immunity .

What expression systems yield optimal production of functional recombinant P. syringae pv. tomato eno1?

Selecting the appropriate expression system for recombinant P. syringae pv. tomato eno1 depends on research objectives and downstream applications:

• Escherichia coli expression systems:

  • BL21(DE3) strains typically yield high quantities of eno1 with good solubility

  • Codon optimization may improve expression efficiency of P. syringae genes in E. coli

  • IPTG-inducible systems like pET vectors allow controlled expression

  • Fusion tags (His6, GST, MBP) facilitate purification and can enhance solubility

• Yeast expression systems:

  • Pichia pastoris or Saccharomyces cerevisiae systems provide eukaryotic post-translational processing

  • Useful when bacterial expression results in insoluble or incorrectly folded protein

  • Commercial platforms (shown in search results) use yeast expression for some recombinant proteins

• Pseudomonas-based expression:

  • Homologous expression in P. fluorescens Pf0-1 using techniques like those described for recombineering

  • May preserve native folding and modifications

  • Allows for studies in a genetic background similar to the native organism

• Purification strategies:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Size exclusion chromatography to achieve >85% purity

  • Buffer optimization to maintain stability during storage (50% glycerol in Tris-based buffer is common)

Recommended protocol elements based on commercial recombinant production include storage at -20°C/-80°C for extended shelf life and avoiding repeated freeze-thaw cycles .

How can researchers validate the functional activity of recombinant P. syringae pv. tomato eno1?

Comprehensive validation of recombinant eno1 functionality requires multiple analytical approaches:

• Enzymatic activity assays:

  • Spectrophotometric assay monitoring conversion of 2-phosphoglycerate to phosphoenolpyruvate

  • Coupled assay systems linking eno1 activity to NADH oxidation for continuous monitoring

  • Michaelis-Menten kinetics determination (Km, Vmax, kcat)

  • Comparison of kinetic parameters to those of native enzyme in crude P. syringae extracts

• Structural validation:

  • Circular dichroism to confirm secondary structure integrity

  • Thermal shift assays to assess protein stability

  • Dynamic light scattering to confirm monodispersity

  • Limited proteolysis to assess proper folding

• Functional characterization:

  • Temperature and pH profile determination

  • Metal ion dependency analysis (Mg2+ is typically required for enolase activity)

  • Substrate specificity testing

  • Inhibition studies with known enolase inhibitors

• Quality control metrics:

  • SDS-PAGE with densitometry to confirm >85% purity

  • Western blot confirmation using tag-specific or eno1-specific antibodies

  • Mass spectrometry to verify the complete amino acid sequence

  • Endotoxin testing for applications requiring high purity

For applications investigating potential non-canonical functions, additional validation may be necessary, such as surface plasmon resonance to confirm binding to suspected interaction partners or cell-based assays to verify biological activity.

What strategies can address challenges in recombinant eno1 expression and purification?

Researchers frequently encounter several challenges when working with recombinant P. syringae proteins. Based on established methodologies in bacterial protein expression, the following troubleshooting approaches are recommended:

• Addressing insolubility:

  • Lower induction temperature (16-25°C) to slow protein folding

  • Reduce inducer concentration for decreased expression rate

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Use solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • Screen multiple buffer conditions during purification

• Improving yield:

  • Optimize codon usage for expression host

  • Test multiple promoter systems (T7, tac, araBAD)

  • Evaluate different E. coli strains (BL21, C41/C43, Arctic Express)

  • Optimize media composition and fermentation conditions

  • Consider auto-induction media for high-density culture

• Enhancing purity:

  • Implement two-step purification strategy (e.g., IMAC followed by gel filtration)

  • Include reducing agents to prevent disulfide-mediated aggregation

  • Add nucleases to remove contaminating nucleic acids

  • Use ion exchange chromatography for charged contaminants

  • Optimize imidazole gradient for His-tagged proteins

• Maintaining stability:

  • Screen buffer additives (glycerol, reducing agents, metal ions)

  • Determine optimal pH and ionic strength

  • Use differential scanning fluorimetry to assess thermal stability

  • Aliquot and flash-freeze to avoid freeze-thaw cycles

  • Consider lyophilization for long-term storage

These approaches have been successfully applied to express and purify various P. syringae proteins, including type III secretion system components and effector proteins , and can be adapted for eno1 expression.

How might recombinant eno1 contribute to understanding P. syringae pv. tomato evolutionary dynamics?

Recombinant eno1 can serve as a valuable tool for investigating evolutionary aspects of P. syringae pv. tomato:

• Comparative evolutionary analysis:

  • Using recombinant eno1 proteins from different P. syringae pathovars to study functional conservation

  • Comparing enzymatic properties of eno1 from race 0 versus race 1 strains

  • Investigating whether metabolic enzymes show adaptation to specific host environments

• Molecular evolution studies:

  • Unlike T3SEs that show extensive evidence of horizontal gene transfer and positive selection , core metabolic genes like eno1 typically evolve under purifying selection

  • Sequence analysis of eno1 across the P. syringae species complex (590+ genomes) could identify subtle adaptive signatures

  • Recombinant proteins from different lineages allow functional testing of any observed sequence variations

• Phylogenomic applications:

  • As a conserved housekeeping gene, eno1 can serve as a phylogenetic marker

  • Comparing eno1 phylogeny with whole-genome phylogeny can reveal instances of horizontal gene transfer

  • Analysis of synonymous versus non-synonymous substitution rates can identify selection pressures

Recent research has demonstrated that P. syringae has a large pangenome of 77,728 orthologous gene families, with a core genome of only 2,410 genes . Investigating how conserved metabolic enzymes like eno1 contribute to adaptation within this diverse species complex could provide insights into bacterial evolution and host specialization.

What role might eno1 play in P. syringae pv. tomato host adaptation and virulence?

While type III secreted effectors are the primary determinants of host specificity in P. syringae , metabolic enzymes like eno1 may contribute to adaptive fitness in specific host environments:

• Potential roles in host adaptation:

  • Optimization of carbon metabolism for growth in tomato apoplastic environment

  • Possible non-canonical functions in biofilm formation or stress responses

  • Potential interaction with host proteins to facilitate infection

• Comparative approaches:

  • Analysis of eno1 expression during infection of different host plants

  • Comparison of eno1 sequence and function between tomato-adapted strains and strains adapted to other hosts

  • Investigation of whether eno1 variants affect host range or virulence in diverse P. syringae pathovars

• Integration with virulence mechanisms:

  • P. syringae pv. tomato virulence depends primarily on the type III secretion system and its effectors

  • Metabolic adaptation is likely complementary to these specialized virulence systems

  • Studies in other pathogens suggest metabolic enzymes may have moonlighting functions in virulence

The molecular basis of P. syringae pv. tomato pathogenesis involves multiple factors beyond T3SEs, including toxins, extracellular proteins, and polysaccharides . Understanding how central metabolic enzymes like eno1 integrate with these virulence systems represents an emerging research direction.

How can recombinant eno1 be utilized to develop novel control strategies for bacterial speck disease?

Recombinant P. syringae pv. tomato eno1 offers several potential pathways toward disease control strategies:

• Target-based inhibitor development:

  • High-throughput screening of chemical libraries against recombinant eno1

  • Structure-based design of inhibitors targeting P. syringae eno1

  • Evaluation of inhibitor specificity between bacterial and plant enolases

  • In vitro and in planta testing of candidate inhibitors

• Immunological approaches:

  • Using recombinant eno1 to develop antibodies for diagnostic applications

  • Exploration of eno1 as a potential vaccine antigen for plant immunization strategies

  • Investigation of whether external application of eno1 can trigger plant immune responses

• Biocontrol development:

  • Screen for microbial antagonists that inhibit eno1 function

  • Engineer non-pathogenic bacteria to express eno1-targeting molecules

  • Explore whether bacteriophages (like those described for P. syringae biocontrol ) can be modified to target eno1-dependent processes

• Integration with current control methods:

  • Combine with effector-triggered immunity (ETI) approaches, which have shown promise as biocontrol agents

  • Assess synergistic effects with copper-based bactericides or other treatments

  • Develop multi-target strategies addressing both specialized virulence factors and metabolic vulnerabilities

Recent research has shown that ETI-eliciting effectors can protect tomato against P. syringae infection when delivered by non-virulent strains . Similar approaches could potentially incorporate strategies targeting metabolic vulnerabilities through eno1 inhibition.

How do post-translational modifications affect eno1 function in P. syringae pv. tomato?

Post-translational modifications (PTMs) of bacterial enolases represent an emerging research area with implications for both metabolic and non-metabolic functions:

• Potential PTMs in bacterial enolases:

  • Phosphorylation affecting catalytic activity or protein interactions

  • Acetylation modulating subcellular localization

  • S-nitrosylation responding to nitrosative stress

  • Oxidative modifications under stress conditions

• Methodological approaches:

  • Mass spectrometry-based proteomics to identify and map PTMs

  • Site-directed mutagenesis of modified residues in recombinant eno1

  • Comparison of PTM patterns between growth in culture and during plant infection

  • In vitro enzymatic assays to assess functional consequences of modifications

• Technical considerations:

  • Expression system selection affects PTM fidelity (bacterial vs. yeast)

  • Specialized purification protocols may be needed to preserve labile modifications

  • Antibodies recognizing specific PTMs could enable targeted studies

This research direction represents a frontier in understanding bacterial protein regulation during host interaction, potentially revealing new dimensions of P. syringae adaptation to plant environments.

How might comparative analysis of eno1 across P. syringae pathovars inform host specificity mechanisms?

The P. syringae species complex includes pathovars with diverse host ranges. Comparative analysis of eno1 across these pathovars could yield insights into metabolic adaptation to different plant hosts:

• Across-pathovar comparison approaches:

  • Recombinant expression of eno1 from multiple pathovars (pv. tomato, pv. syringae, pv. actinidiae, etc.)

  • Comparative biochemical characterization (kinetic parameters, pH optima, stability)

  • Cross-complementation experiments in different pathovar backgrounds

  • Computational analysis of sequence conservation patterns

• Host adaptation signatures:

  • Analysis of selection pressure on eno1 in different host-specialized lineages

  • Identification of pathovar-specific amino acid substitutions

  • Correlation of sequence variations with biochemical properties and infection phenotypes

• Integration with genomic data:

  • Recent genomic studies have characterized P. syringae strains from diverse hosts

  • Correlation of eno1 variants with core genome phylogeny

  • Investigation of potential co-evolution with other metabolic or virulence genes

While effector repertoires are primary determinants of host range , metabolic adaptations may contribute to optimal fitness in specific host environments. Comparative analysis could reveal subtle patterns of metabolic specialization across the P. syringae complex.

What interdisciplinary approaches could reveal novel insights about eno1 in plant-pathogen interactions?

Integrating multiple disciplines and technologies could drive breakthroughs in understanding eno1's role in P. syringae biology and pathogenicity:

• Systems biology integration:

  • Metabolic modeling of P. syringae during infection with variation in eno1 function

  • Network analysis positioning eno1 in both metabolic and virulence-related pathways

  • Multi-omics approaches correlating transcriptomics, proteomics, and metabolomics data

• Structural biology and protein dynamics:

  • Cryo-EM or X-ray crystallography of P. syringae eno1 alone and in protein complexes

  • Molecular dynamics simulations to investigate conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

• Advanced microscopy techniques:

  • Super-resolution microscopy tracking eno1 localization during infection

  • Correlative light and electron microscopy to place eno1 in ultrastructural context

  • FRET-based biosensors to monitor eno1 activity in living bacteria

• Synthetic biology approaches:

  • Engineering P. syringae with modified eno1 variants to test specific hypotheses

  • Creating minimal systems to reconstitute eno1-dependent phenomena

  • Using recombineering techniques similar to those described for P. syringae to introduce precise genomic modifications

These interdisciplinary approaches could position eno1 research within the broader context of plant-pathogen interaction biology, potentially revealing unexpected connections between metabolism and virulence.