Recombinant Xylella fastidiosa Triosephosphate isomerase (tpiA)

What is the role of triosephosphate isomerase (tpiA) in Xylella fastidiosa metabolism?

Triosephosphate isomerase (tpiA) in Xylella fastidiosa functions as a critical enzyme in central carbon metabolism, catalyzing the reversible interconversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GA3P). This isomerization represents an essential step in both glycolysis and gluconeogenesis pathways. Similar to its role in other bacteria like Escherichia coli, tpiA in X. fastidiosa likely plays a crucial role in carbon utilization efficiency, particularly when the bacterium grows on 3-carbon substrates . The enzyme's function becomes especially important in the xylem environment where X. fastidiosa resides, which is relatively nutrient-poor and requires efficient carbon metabolism for bacterial survival and proliferation. The conservation of this enzyme across bacterial species underscores its fundamental metabolic importance.

Is tpiA essential for X. fastidiosa growth and virulence in planta?

Based on growth studies in model organisms like E. coli, where tpiA deletion abolishes growth on glycerol but not glucose , we can hypothesize that tpiA essentiality in X. fastidiosa would depend on carbon source availability within plant xylem tissues. The methodological approach to test this would include:

• Generation of tpiA knockout mutants in X. fastidiosa (challenging due to transformation difficulties)

• Comparative growth analysis in defined media with different carbon sources

• Plant inoculation experiments comparing wild-type and tpiA mutant strains

• Measurement of bacterial proliferation, movement, and symptom development

This essentiality question is particularly relevant because X. fastidiosa causes impaired water movement in infected plants , and metabolic adaptations may directly influence pathogenicity mechanisms. Researchers should complement genetic approaches with metabolomic analyses to fully understand the relationship between tpiA function and virulence in different plant hosts.

What expression systems are optimal for recombinant X. fastidiosa tpiA production?

When selecting expression systems for recombinant X. fastidiosa tpiA, researchers must consider several factors affecting protein folding, solubility, and activity. Based on successful approaches with similar enzymes, a methodological workflow would include:

• Vector selection and construct design:

  • pET-based expression systems with T7 promoter for high expression

  • Addition of affinity tags (His6, E-tag) positioned to avoid interference with enzyme activity

  • Inclusion of TEV protease cleavage sites for tag removal if required

  • Optional fusion partners (SUMO, MBP) to enhance solubility if expression yields are low

• Host strain evaluation:

  • E. coli BL21(DE3) as primary expression host

  • BL21(DE3)pLysS for potential toxic effects

  • Rosetta or CodonPlus strains if codon usage analysis indicates rare codons

  • Consideration of chaperone co-expression systems as tpiA appears to interact with DnaK in some bacteria

• Expression condition optimization:

  • Systematic testing of induction temperatures (16°C, 25°C, 37°C)

  • IPTG concentration titration (0.1-1.0 mM)

  • Evaluation of expression duration (4h vs. overnight)

Since tpiA is highly conserved and typically soluble, heterologous expression in E. coli should yield active enzyme, but validation against native X. fastidiosa tpiA activity is essential to confirm proper folding.

What purification strategies maximize yield and activity of recombinant X. fastidiosa tpiA?

Purification of recombinant X. fastidiosa tpiA requires a balance between yield, purity, and retention of enzymatic activity. A comprehensive purification protocol would include:

• Initial extraction and clarification:

  • Cell lysis by sonication or high-pressure homogenization in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol

  • Addition of protease inhibitors to prevent degradation

  • Centrifugation at 20,000×g to remove cellular debris

• Affinity chromatography:

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

  • Immunoaffinity purification for E-tagged versions

  • Gradient elution to separate differentially binding contaminants

• Secondary purification:

  • Ion exchange chromatography based on theoretical pI of X. fastidiosa tpiA

  • Size exclusion chromatography to ensure oligomeric homogeneity and remove aggregates

• Activity preservation strategies:

  • Addition of stabilizing agents (glycerol 10-20%)

  • Evaluation of optimal buffer conditions for stability

  • Determination of appropriate storage conditions (-80°C vs. liquid nitrogen)

After each purification step, researchers should verify tpiA activity using standard assays as described in section 3.1, comparing activity per mg protein to monitor purification efficiency and recovery.

How can researchers verify the correct folding and activity of recombinant X. fastidiosa tpiA?

Verification of correct folding and activity is crucial for reliable experimental outcomes with recombinant X. fastidiosa tpiA. A multi-technique approach should include:

• Enzymatic activity assays:

  • Spectrophotometric coupled assay with α-glycerophosphate dehydrogenase and NADH

  • Determination of specific activity (μmol/min/mg protein)

  • Comparison with reference enzymes (e.g., commercially available tpiA)

• Structural integrity analysis:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Thermal denaturation studies to determine melting temperature (Tm)

  • Limited proteolysis to probe for compact, well-folded structure

• Oligomeric state verification:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Native PAGE analysis

  • Analytical ultracentrifugation for detailed quaternary structure assessment

• Functional complementation:

  • Transformation of an E. coli ΔtpiA strain with the recombinant X. fastidiosa tpiA

  • Growth assessment on minimal medium with glycerol as sole carbon source

  • Comparison of growth rates between complemented strain and wild-type

A properly folded and active recombinant X. fastidiosa tpiA should exhibit expected enzymatic parameters, maintain native-like structural features, and functionally complement a tpiA-deficient bacterial strain.

What are the standard assays for measuring X. fastidiosa tpiA enzyme activity?

Several well-established assays can be adapted for X. fastidiosa tpiA activity measurement, with the coupled spectrophotometric assay being the gold standard. The methodological approach includes:

• Coupled spectrophotometric assay:

  • Principle: Coupling tpiA-catalyzed isomerization to NADH oxidation via α-glycerophosphate dehydrogenase

  • Reaction mixture: 100 mM Tris-HCl (pH 7.5), 1 mM dihydroxyacetone phosphate (DHAP), 0.2 mM NADH, excess α-glycerophosphate dehydrogenase

  • Measurement: Decrease in absorbance at 340 nm as NADH is oxidized

  • Calculation: Activity (μmol/min) = ΔA340/min ÷ 6.22 (extinction coefficient of NADH)

• Direct product analysis:

  • HPLC or mass spectrometry-based quantification of DHAP and GA3P interconversion

  • Requires separation of reactants and products on appropriate columns

  • Provides direct measurement without coupling to secondary reactions

• In vivo complementation assay:

  • Quantitative assessment of growth rates of E. coli ΔtpiA strain expressing X. fastidiosa tpiA

  • Growth curve analysis in minimal medium with glycerol as sole carbon source

  • Comparison with positive control (native tpiA) and negative control (empty vector)

When performing these assays, researchers should generate standard curves of activity versus enzyme concentration to ensure linearity and determine the appropriate enzyme concentration ranges for kinetic studies.

How do environmental factors affect X. fastidiosa tpiA activity and stability?

Understanding environmental factors affecting X. fastidiosa tpiA is crucial given the variability in conditions across different plant hosts and geographic regions where this pathogen occurs. The methodological approach includes:

• Temperature dependence studies:

  • Measurement of enzymatic activity across temperature range (15-45°C)

  • Determination of temperature optimum and activation energy

  • Thermal stability assessment through pre-incubation at various temperatures

  • Correlation with growth temperature ranges of X. fastidiosa in different geographical regions

• pH profile analysis:

  • Activity measurements across pH range (5.0-9.0) using appropriate buffer systems

  • Construction of pH-activity profile identifying optimum pH

  • Analysis of pH effects on enzyme kinetic parameters (Km, Vmax)

  • Correlation with xylem pH in different host plants

• Ion effects evaluation:

  • Systematic testing of monovalent and divalent cations (Na+, K+, Mg2+, Ca2+)

  • Assessment of potential inhibitors present in plant xylem

  • Metal chelator effects to identify potential metal cofactor requirements

These studies should be conducted with purified enzyme and validated in more complex systems that better mimic plant xylem conditions. Results can provide insights into how X. fastidiosa tpiA function may vary across different plant hosts and environmental conditions, potentially contributing to host specificity patterns observed among X. fastidiosa strains .

How does strain variation affect tpiA enzyme properties among X. fastidiosa subspecies?

X. fastidiosa exists as several subspecies with different host ranges and virulence characteristics . Investigation of tpiA variation across these strains may reveal adaptations to specific hosts or environments. The methodological approach includes:

• Comparative sequence analysis:

  • Multiple sequence alignment of tpiA genes from diverse X. fastidiosa strains

  • Identification of subspecies-specific amino acid substitutions

  • Phylogenetic analysis correlating tpiA sequence clusters with host specificity

  • Structural mapping of variable residues onto 3D models

• Recombinant expression of variant enzymes:

  • Cloning and expression of tpiA from representative strains of each subspecies

  • Standardized purification and activity determination

  • Comparative kinetic characterization (Km, kcat, substrate specificity)

• Environmental response comparison:

  • Side-by-side testing of subspecies variants under various conditions

  • Construction of activity profiles under different temperature, pH, and ionic conditions

  • Statistical analysis to identify significant differences between variants

This cross-subspecies analysis can provide valuable insights into how metabolic adaptations may contribute to host specialization and virulence differences among X. fastidiosa strains.

What strategies overcome the challenges of genetic manipulation in X. fastidiosa?

Genetic manipulation of X. fastidiosa presents significant challenges due to transformation difficulties and restriction-modification systems . For tpiA studies, researchers should consider these methodological approaches:

• Overcoming restriction barriers:

  • Methylation analysis of X. fastidiosa genome to identify restriction patterns

  • Pre-methylation of recombinant DNA using appropriate methyltransferases

  • Use of plasmids isolated from X. fastidiosa itself to match endogenous methylation patterns

  • Transformation into restriction-deficient X. fastidiosa strains if available

• Transformation optimization:

  • Electroporation with customized parameters (field strength, pulse duration)

  • Natural competence induction under specific growth conditions

  • Use of broad host-range plasmids with origins functional in X. fastidiosa

  • Recovery in specialized media optimized for post-transformation survival

• Gene editing approaches:

  • Suicide vector strategies with counter-selectable markers

  • Homologous recombination with extended homology arms (>1 kb)

  • CRISPR-Cas9 systems adapted for X. fastidiosa

  • Recombineering approaches if lambda Red-like systems can be established

• Verification strategies:

  • PCR confirmation of genetic modifications

  • Whole genome sequencing to verify desired changes and exclude off-target effects

  • Phenotypic validation through appropriate growth and activity assays

  • RNA-seq to confirm expression changes in modified strains

Researchers should note that type I restriction-modification systems in X. fastidiosa demonstrate significant diversity in target recognition domains across strains , necessitating strain-specific optimization of genetic manipulation protocols.

How can researchers develop tpiA mutants to study metabolic pathways in X. fastidiosa?

Creating tpiA mutants in X. fastidiosa requires careful consideration of this enzyme's central metabolic role. The methodological approach includes:

• Knockout strategy design:

  • Complete gene deletion using homologous recombination

  • Domain-specific mutations targeting catalytic residues

  • Conditional expression systems if tpiA proves essential

  • Partial activity mutants through targeted amino acid substitutions

• Complementation systems:

  • Trans-complementation with wild-type tpiA under native or inducible promoters

  • Metabolic bypass strategies providing alternative carbon processing routes

  • Heterologous tpiA expression from other species to assess functional conservation

• Phenotypic characterization:

  • Growth analysis on defined media with various carbon sources

  • Metabolomic profiling to identify pathway shifts and metabolite accumulation

  • In planta studies to assess virulence and colonization capacity

  • Transcriptomic analysis to identify compensatory responses

• Specialized techniques for essential gene studies:

  • CRISPR interference (CRISPRi) for tunable repression if knockouts are lethal

  • Degradation tag systems for controlled protein depletion

  • Temperature-sensitive alleles if they can be developed

Given that tpiA deletion in E. coli prevents growth on glycerol but not glucose , researchers should design media conditions carefully to allow for viable mutant isolation while confirming the expected phenotypes.

How does the type I restriction-modification system in X. fastidiosa affect recombinant tpiA studies?

The type I restriction-modification (R-M) systems in X. fastidiosa represent a significant consideration for recombinant DNA studies . For tpiA research, addressing these systems involves:

• R-M system characterization:

  • Identification of active R-M systems in the specific X. fastidiosa strain

  • Analysis of target recognition domains (TRDs) and their recognition sequences

  • Methylome analysis to map genome-wide methylation patterns

  • Quantification of restriction enzyme activity in cellular extracts

• Overcoming restriction barriers:

  • In vitro methylation of recombinant DNA prior to transformation

  • Use of plasmids isolated from the same X. fastidiosa strain

  • Engineering recombinant DNA to eliminate recognized restriction sites

  • Temporary inactivation of R-M systems if technically feasible

• Strain-specific considerations:

  • X. fastidiosa strains possess 31 different allele profiles for R-M systems

  • Some strains contain inactivating mutations in type I R-M systems

  • Selection of transformation-amenable strains for initial studies

  • Transfer of validated constructs to more restrictive strains using appropriate strategies

Understanding and addressing the R-M systems is particularly important for tpiA studies requiring gene complementation or expression of recombinant variants, as transformation efficiency can vary dramatically based on DNA methylation status.

How does tpiA activity relate to X. fastidiosa virulence and plant colonization?

Understanding the connection between central metabolism and virulence is crucial for X. fastidiosa pathogenesis research. Methodological approaches to investigate this relationship include:

• Correlation studies:

  • Measurement of tpiA expression during different stages of plant infection

  • Comparative analysis of tpiA activity across strains with different virulence levels

  • Metabolic flux analysis to determine glycolytic activity during colonization

  • Correlation of tpiA activity with bacterial population growth in planta

• Manipulation experiments:

  • Creation of tpiA variants with altered activity levels

  • Plant inoculation with modified strains to assess colonization efficiency

  • Monitoring of disease symptom development and progression

  • Quantification of bacterial movement through plant vasculature

• Mechanism investigation:

  • Analysis of metabolic byproducts that may act as virulence factors

  • Investigation of potential moonlighting functions of tpiA beyond metabolism

  • Assessment of tpiA role in stress resistance during plant colonization

  • Evaluation of tpiA's contribution to biofilm formation in xylem vessels

These approaches can help determine whether tpiA plays a direct role in virulence or if its effects are mediated through general metabolism. This is particularly relevant given that X. fastidiosa impairs water movement in infected plants , which could potentially involve metabolically-derived compounds or biofilms dependent on central carbon metabolism.

Can recombinant X. fastidiosa tpiA be used to develop diagnostic or control strategies?

Leveraging recombinant tpiA for applied purposes represents an important research direction. Methodological approaches include:

• Diagnostic application development:

  • Generation of anti-tpiA antibodies for immunodetection systems

  • Development of activity-based assays for early detection

  • Creation of biosensors incorporating tpiA-specific aptamers

  • Integration with thermal imaging approaches for pre-symptomatic detection

• Control strategy exploration:

  • High-throughput screening for specific tpiA inhibitors

  • Structure-based drug design targeting X. fastidiosa-specific features

  • Evaluation of inhibitor specificity against plant and beneficial microbe tpiA

  • In planta testing of promising inhibitory compounds

• Vaccination-like approaches:

  • Development of tpiA-based preparations to trigger plant immune responses

  • Creation of inactive tpiA variants that could prime defense without pathogenesis

  • Evaluation of systemic acquired resistance induction by tpiA-derived peptides

• Engineering resistance:

  • Identification of plant proteins interacting with bacterial tpiA

  • Generation of decoy molecules to disrupt potential tpiA interactions

  • Engineering of plant factors that specifically inhibit X. fastidiosa tpiA

These applications become particularly relevant considering the challenges in detecting X. fastidiosa during latent infection periods and the significant economic impacts of diseases like Pierce's disease, olive quick decline syndrome, and citrus variegated chlorosis .

How can tpiA be used to study host adaptation in different X. fastidiosa subspecies?

The diverse host range of X. fastidiosa subspecies provides an opportunity to investigate metabolic adaptations in host specialization. For tpiA studies, methodological approaches include:

• Comparative enzymatic analysis:

  • Expression and purification of tpiA from multiple subspecies

  • Side-by-side kinetic characterization under standardized conditions

  • Determination of substrate affinities and catalytic efficiencies

  • Assessment of performance under conditions mimicking different host xylem environments

• Host-specific expression studies:

  • Quantification of tpiA expression levels in different plant hosts

  • Analysis of potential post-translational modifications in different host environments

  • Investigation of regulatory mechanisms controlling tpiA expression

  • Correlation of expression patterns with bacterial growth rates in specific hosts

• Cross-complementation experiments:

  • Expression of tpiA from one subspecies in another subspecies background

  • Assessment of growth, metabolism, and virulence in the native host of recipient strain

  • Identification of subspecies-specific features that cannot be complemented

  • Construction of chimeric enzymes to pinpoint functionally distinct regions

These studies can reveal whether metabolic enzyme adaptations contribute to host range determination in X. fastidiosa, complementing known factors like adhesion and motility.

What structural features of X. fastidiosa tpiA might contribute to strain-specific metabolism?

Advanced structural biology approaches can reveal subtle adaptations in X. fastidiosa tpiA that may influence metabolism in different strains. The methodological approach includes:

• High-resolution structural determination:

  • X-ray crystallography of purified recombinant tpiA from representative strains

  • Cryo-electron microscopy for challenging variants

  • NMR studies of dynamics and substrate interactions

  • Computational modeling validated by experimental data

• Structural comparison across subspecies:

  • Superposition analysis to identify subspecies-specific structural differences

  • Molecular dynamics simulations to assess flexibility and conformational sampling

  • Calculation of electrostatic surface potentials to identify interaction differences

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

• Structure-function correlation:

  • Site-directed mutagenesis targeting subspecies-specific residues

  • Activity measurements with natural and artificial substrates

  • Thermal stability comparisons across variants

  • Protein-protein interaction studies to identify potential metabolic partners

• Engineering applications:

  • Rational design of chimeric enzymes with defined substrate preferences

  • Creation of reporter constructs based on structural insights

  • Development of structure-based inhibitors with subspecies specificity

Drawing from observations in E. coli tpiA, where structural robustness allows for significant insertions without loss of function , researchers should investigate whether X. fastidiosa tpiA exhibits similar structural permissiveness and how this might contribute to evolutionary adaptability.

How might epigenetic modifications affect tpiA expression across X. fastidiosa strains?

The diversity of methylation patterns associated with type I restriction-modification systems in X. fastidiosa raises questions about potential epigenetic regulation of tpiA. The methodological approach includes:

• Methylome analysis:

  • Bisulfite sequencing or SMRT sequencing to map genome-wide methylation patterns

  • Targeted analysis of tpiA promoter regions across strains

  • Correlation of methylation patterns with tpiA expression levels

  • Comparison of methylation at regulatory elements across subspecies

• Experimental manipulation:

  • Methyltransferase overexpression or inhibition to alter methylation patterns

  • Reporter constructs with native and modified promoter regions

  • CRISPR-dCas9 approaches to target epigenetic modifiers to specific locations

  • Correlation of induced methylation changes with expression alterations

• Regulatory network analysis:

  • Identification of transcription factors binding to the tpiA promoter

  • Investigation of methylation effects on transcription factor binding

  • Analysis of small RNA regulation potentially affected by methylation

  • Integration of epigenetic data with transcriptomic and proteomic datasets

This research area connects tpiA regulation with the broader question of how type I R-M systems influence gene expression patterns and potentially contribute to phenotypic diversity across X. fastidiosa strains .

What emerging technologies can advance X. fastidiosa tpiA research in pathogenesis?

Cutting-edge methodologies offer new opportunities to understand tpiA's role in X. fastidiosa biology and pathogenesis. Promising approaches include:

• Single-cell technologies:

  • Single-cell RNA-seq to capture expression heterogeneity during infection

  • Time-lapse microscopy with fluorescent tpiA reporters

  • Microfluidic platforms to study metabolic responses at the single-cell level

  • Correlation of tpiA activity with individual cell behaviors in planta

• Advanced imaging techniques:

  • Super-resolution microscopy to localize tpiA within bacterial cells

  • Thermal imaging for early detection of metabolic changes in infected plants

  • Raman spectroscopy to track metabolic shifts non-invasively

  • Molecular imaging of tpiA activity using specialized probes

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Genome-scale metabolic modeling with tpiA flux constraints

  • Network analysis to position tpiA in the broader context of metabolism and virulence

  • Machine learning applications to identify subtle patterns in tpiA-related datasets

• In situ techniques:

  • FISH-based detection of tpiA expression in plant tissues

  • Laser capture microdissection combined with molecular analysis

  • In planta CRISPR interference to modulate tpiA expression during infection

  • Direct enzyme activity measurements in extracted xylem sap

These technologies, particularly when combined, can provide unprecedented insights into how tpiA functions within the complex environment of infected plant tissue, potentially revealing new intervention points for disease management.