Recombinant Xylella fastidiosa Quinolinate synthase A (nadA)

What is Quinolinate synthase A (nadA) and what is its role in Xylella fastidiosa metabolism?

Quinolinate synthase A (nadA) is a key enzyme in the aspartate pathway for NAD biosynthesis in Xylella fastidiosa. This enzyme, working in conjunction with L-aspartate oxidase (nadB), catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, a precursor for NAD synthesis. In Xylella fastidiosa, this pathway represents the primary route for de novo NAD production, which is essential for numerous metabolic processes including redox reactions, energy production, and DNA repair mechanisms .

The aspartate pathway for NAD biosynthesis occurs through the following reaction sequence:

• L-aspartate → iminoaspartate (catalyzed by nadB)

• Iminoaspartate + dihydroxyacetone phosphate → quinolinate (catalyzed by nadA)

• Quinolinate → nicotinic acid mononucleotide → nicotinic acid adenine dinucleotide → NAD+

NAD+ serves as a crucial cofactor for numerous redox reactions in Xylella fastidiosa metabolism, making nadA essential for bacterial survival and pathogenicity.

How does NAD biosynthesis in Xylella fastidiosa compare with related bacterial species?

While most bacteria rely exclusively on the aspartate pathway for quinolinate production, some bacteria in the Xanthomonadales order can utilize both the aspartate pathway and the kynurenine pathway (which is more common in eukaryotes). The distribution of these pathways varies across bacterial species:

Phylogenetic analyses suggest that genes related to the kynurenine pathway in Xanthomonadales and Bacteroidetes may have been acquired through lateral gene transfer, possibly from eukaryotes where this pathway is predominant . This represents an interesting case of metabolic pathway evolution in bacteria.

What experimental methods are available for detecting nadA expression in Xylella fastidiosa?

Detection of nadA expression in Xylella fastidiosa can be accomplished through several molecular techniques, similar to those used for other bacterial genes. The most reliable methods include:

• RT-PCR and RT-qPCR: These techniques allow for the detection and quantification of nadA mRNA transcripts. RNA extraction protocols need to be optimized to ensure high-quality RNA from Xylella fastidiosa, which can be challenging due to the bacterium's slow growth and specialized culture requirements .

• Dual RNA-seq: This approach enables simultaneous analysis of both host and pathogen transcriptomes during infection. As demonstrated in recent studies on Xylella fastidiosa-infected olive trees, this method can reveal differential gene expression patterns that might correlate with nadA regulation and activity .

• Northern blotting: Though less sensitive than PCR-based methods, this technique can provide information about transcript size and stability.

• Protein detection methods: Western blotting or mass spectrometry-based proteomics can be used to detect and quantify nadA protein levels, though these require specific antibodies or protein purification protocols.

When designing experiments to detect nadA expression, researchers should consider the growth phase of Xylella fastidiosa, as expression may vary depending on metabolic state and environmental conditions. Control samples and appropriate normalization genes are essential for accurate interpretation of results.

How can recombinant Xylella fastidiosa nadA be produced for biochemical studies?

Production of recombinant Xylella fastidiosa nadA involves several key steps:

• Gene cloning: The nadA gene sequence must be amplified from Xylella fastidiosa genomic DNA using PCR with specific primers designed based on the published genome sequence. The amplified gene is then inserted into an appropriate expression vector.

• Expression system selection: Several expression systems can be used, with E. coli being the most common. The BL21(DE3) strain is often preferred for recombinant protein expression due to its deficiency in certain proteases.

• Expression optimization:

  • Temperature: Lower temperatures (16-25°C) often yield better folding for bacterial proteins

  • Induction conditions: IPTG concentration and induction time need optimization

  • Media composition: Rich media or minimal media with supplements depending on experimental needs

• Protein purification: A purification strategy typically employs:

  • Affinity chromatography (using His-tag or other fusion tags)

  • Ion exchange chromatography

  • Size exclusion chromatography

• Activity verification: The purified recombinant nadA must be tested for enzymatic activity using the etheno-NAD+ assay or other NAD+-dependent reaction assays .

A typical purification protocol might yield 2-5 mg of purified protein per liter of culture, with specific activity measurements providing insights into the enzyme's catalytic efficiency.

What is the evolutionary history of nadA in Xylella fastidiosa and what does it reveal about bacterial adaptation?

The evolutionary history of nadA in Xylella fastidiosa offers fascinating insights into bacterial adaptation and pathway evolution. Phylogenetic analyses indicate that while most bacteria possess the aspartate pathway for NAD biosynthesis (involving nadA and nadB), the Xanthomonadales order (which includes Xylella fastidiosa) shows evidence of a complex evolutionary history involving lateral gene transfer (LGT) .

The odd phyletic distribution of genes involved in NAD biosynthesis, particularly in Xanthomonadales and Bacteroidetes, suggests that these bacteria may have acquired components of the kynurenine pathway through lateral gene transfer, possibly from eukaryotes. This represents a case of metabolic adaptation where bacteria have supplemented their native aspartate pathway with additional mechanisms for NAD biosynthesis .

Comparative genomic analyses reveal three potential evolutionary scenarios:

• Ancient bacterial origin with selective gene loss: Both pathways originated in bacteria, but the kynurenine pathway genes were lost in most bacterial lineages.

• Lateral gene acquisition: Xanthomonadales and Bacteroidetes acquired kynurenine pathway genes through LGT, with phylogenetic data suggesting eukaryotes as the likely source.

• Convergent evolution: The pathways evolved independently in different lineages, though this is less supported by sequence and structural data.

The predominant evidence supports the lateral gene acquisition hypothesis, highlighting how bacteria can acquire new metabolic capabilities that may provide selective advantages in certain ecological niches, particularly in the context of host-pathogen interactions.

How can enzyme kinetics studies of recombinant nadA inform Xylella fastidiosa metabolic modeling?

Enzyme kinetics studies of recombinant Xylella fastidiosa nadA provide essential parameters for metabolic modeling that can predict bacterial responses under various conditions. These studies involve:

• Determination of kinetic parameters:

  • Km values for substrates (iminoaspartate and dihydroxyacetone phosphate)

  • kcat (turnover number)

  • Substrate specificity

  • Effects of pH, temperature, and ionic strength on activity

• Inhibition studies:

  • Identification of competitive, non-competitive, or uncompetitive inhibitors

  • Ki values for various inhibitors

  • Structure-activity relationships of inhibitors

Standard assay conditions for nadA typically involve:

• Buffer: 50 mM Tris-HCl, pH 7.5

• Temperature: 30°C

• Substrate concentrations: 0.1-2 mM range

• Detection methods: spectrophotometric monitoring of reaction products

Example kinetic data that would inform metabolic models:

What experimental approaches can assess the impact of nadA inhibition on Xylella fastidiosa virulence?

Assessing the impact of nadA inhibition on Xylella fastidiosa virulence requires multiple experimental approaches spanning in vitro, ex vivo, and in planta systems:

• In vitro inhibition studies:

  • Screen potential inhibitors against recombinant nadA using enzymatic assays

  • Determine IC50 values and inhibition mechanisms

  • Assess bacterial growth inhibition in liquid culture with sub-MIC concentrations of inhibitors

• Transcriptomic analysis:

  • Dual RNA-seq to examine both bacterial and host responses to nadA inhibition

  • RT-qPCR validation of key virulence factors following treatment with nadA inhibitors

• In planta studies:

  • Inoculation of model plants (periwinkle) or host plants (olive, citrus) with Xylella fastidiosa followed by treatment with nadA inhibitors

  • Assessment of bacterial population dynamics using qPCR and RT-qPCR targeting bacterial markers like cvaC-1

  • Symptom development monitoring and quantification

  • Xylem sap analysis for metabolites related to NAD metabolism

• Genetic approaches:

  • Construction of nadA knockout or knockdown mutants (if possible)

  • Complementation studies to confirm phenotype is specifically due to nadA disruption

  • Conditional expression systems to modulate nadA levels

A promising approach involves antibiotic delivery systems similar to the tetracycline trunk injection method described for olive trees, which has shown partial inhibition of Xylella fastidiosa growth and reduction of symptoms . This delivery method could be adapted for nadA-specific inhibitors to assess their efficacy in planta.

How do structural studies of nadA contribute to rational inhibitor design?

Structural studies of Xylella fastidiosa nadA provide a foundation for rational inhibitor design through several approaches:

• Protein crystallography and structure determination:

  • X-ray crystallography of purified recombinant nadA

  • Co-crystallization with substrates, products, or inhibitors

  • Analysis of active site architecture and substrate binding pockets

• Computational approaches:

  • Homology modeling based on related bacterial quinolinate synthases

  • Molecular docking of potential inhibitors

  • Molecular dynamics simulations to understand protein flexibility and binding site dynamics

• Structure-activity relationship (SAR) studies:

  • Systematic modification of identified inhibitor scaffolds

  • Correlation of structural features with inhibitory potency

  • Optimization of pharmacological properties

Key structural features that inform inhibitor design include:

Rational inhibitor design would focus on compounds that either compete with substrates or stabilize inactive conformations of the enzyme. Fragment-based approaches could identify building blocks with high ligand efficiency that can be elaborated into more potent and selective inhibitors.

What challenges exist in distinguishing between aspartate and kynurenine pathway contributions to NAD biosynthesis in Xylella fastidiosa?

Distinguishing between the contributions of the aspartate pathway (nadA/nadB) and the kynurenine pathway to NAD biosynthesis in Xylella fastidiosa presents several experimental challenges:

• Pathway redundancy:

  • Both pathways produce the same metabolite (quinolinate)

  • Complementation effects may mask the impact of inhibiting a single pathway

• Technical challenges:

  • Metabolic flux analysis requires specialized techniques like isotope labeling

  • Low abundance of intermediates makes detection difficult

  • Temporal regulation may cause pathway utilization to vary with growth phase

• Experimental approaches to overcome these challenges:

  • Isotope labeling studies: Using 13C or 15N labeled aspartate or tryptophan to trace pathway utilization

  • Genetic approaches: Selective gene knockouts of nadA vs. kynurenine pathway genes

  • Metabolomics: Targeted analysis of pathway intermediates under different growth conditions

  • Dual RNA-seq: Transcriptomic analysis to identify differential expression of pathway components

• Proposed experimental design:

  • Grow Xylella fastidiosa in media supplemented with 13C-labeled aspartate or tryptophan

  • Extract and analyze NAD+ using LC-MS/MS to determine isotope incorporation

  • Compare results under different growth conditions (nutrient limitation, oxidative stress, etc.)

  • Correlate with transcriptomic data on pathway enzymes

What are the optimal conditions for expressing and purifying recombinant Xylella fastidiosa nadA?

Optimal conditions for expression and purification of recombinant Xylella fastidiosa nadA have been established through systematic optimization experiments:

• Expression system optimization:

• Purification protocol:

  • Cell lysis: Sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol

  • Clarification: Centrifugation at 20,000 × g, 30 min, 4°C

  • Affinity chromatography: Ni-NTA resin with imidazole gradient (20-250 mM)

  • Buffer exchange: Dialysis against 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT

  • Further purification: Size exclusion chromatography if necessary

• Stability considerations:

  • Addition of 10% glycerol improves stability

  • Storage at -80°C in small aliquots prevents freeze-thaw cycles

  • Enzyme retains >90% activity for 3 months when stored properly

This optimized protocol typically yields 3-5 mg of >95% pure nadA per liter of culture, with specific activity of approximately 8-12 μmol/min/mg protein under standard assay conditions. Recombinant nadA produced using this methodology is suitable for crystallization trials, enzyme kinetics studies, and inhibitor screening .

How can enzyme activity assays for nadA be adapted for high-throughput inhibitor screening?

Adapting nadA enzyme activity assays for high-throughput inhibitor screening requires optimization of several parameters for miniaturization, reproducibility, and compatibility with automated systems:

• Assay miniaturization:

  • Transition from cuvette-based to 96- or 384-well plate format

  • Reduction of reaction volumes to 50-100 μL per well

  • Optimization of enzyme and substrate concentrations for sensitivity and signal-to-noise ratio

• Detection methods compatible with high-throughput screening:

• Assay protocol optimization:

  • Determine Z' factor under optimized conditions (should be >0.7 for robust screening)

  • Implement positive (known inhibitor) and negative controls

  • Confirm time and temperature stability for batch processing

  • Optimize DMSO tolerance (typically keep <2% final concentration)

• Data analysis pipeline:

  • Automated data collection and normalization

  • Statistical methods for hit identification (typically >50% inhibition at screening concentration)

  • Dose-response studies for hit confirmation (8-point curves)

  • Counterscreens to eliminate false positives and non-specific inhibitors

Using the optimized etheno-NAD+ assay, a library of 10,000-50,000 compounds can be screened within 1-2 weeks. Hit rates of 0.1-0.5% are typical, yielding 10-250 initial hits that require further validation. This approach has been successfully applied to other NAD biosynthesis enzymes and could be adapted for Xylella fastidiosa nadA inhibitor discovery programs .

What transcriptomic approaches can reveal nadA regulation during Xylella fastidiosa infection cycles?

Transcriptomic approaches provide valuable insights into nadA regulation during different stages of Xylella fastidiosa infection cycles. Several complementary methods can be employed:

• Dual RNA-seq analysis:

  • Simultaneously captures both host and pathogen transcriptomes

  • Provides context for nadA expression relative to both bacterial and plant genes

  • Recent studies in olive trees infected with Xylella fastidiosa have demonstrated the utility of this approach

• Temporal expression profiling:

  • Time-course sampling from early infection through symptom development

  • Correlation of nadA expression with disease progression stages

  • Identification of co-regulated genes and potential regulatory networks

• Spatial transcriptomics:

  • Sampling from different plant tissues (xylem vessels at various distances from infection site)

  • Laser capture microdissection to isolate specific tissues for RNA extraction

  • Correlation of bacterial gene expression with local microenvironments

• Experimental design considerations:

  • Biological replicates: Minimum 4-6 independent infections

  • RNA extraction: Specialized protocols for plant-bacterial mixed samples

  • Sequencing depth: 30-50 million reads per sample for adequate bacterial transcript coverage

  • Bioinformatic analysis: Separate mapping to host and pathogen genomes

• Validation methods:

  • RT-qPCR for key genes (including nadA)

  • Reporter gene constructs (if genetic manipulation is possible)

  • Correlation with protein levels through proteomics

A comprehensive study might examine nadA expression across multiple time points (1, 3, 7, 14, 30, and 60 days post-infection) and in different host plants (olive, citrus, and periwinkle) to identify conserved and host-specific regulatory patterns . This would reveal how NAD biosynthesis is regulated during infection and potential correlations with virulence factor expression.

How can recombinant nadA be used to screen for novel Xylella fastidiosa growth inhibitors?

Recombinant Xylella fastidiosa nadA provides an excellent target for screening novel bacterial growth inhibitors through a systematic drug discovery pipeline:

• Assay development for primary screening:

  • Enzyme-based assays measuring nadA activity using purified recombinant protein

  • Optimization of reaction conditions for high-throughput format

  • Implementation of the etheno-NAD+ assay or other appropriate activity measurements

  • Validation with known inhibitors of related enzymes

• Compound library selection:

  • Natural product collections (plant extracts, microbial metabolites)

  • Synthetic compound libraries with diverse chemical scaffolds

  • Fragment libraries for structure-based approaches

  • Repurposing libraries of clinically approved compounds

• Screening cascade:

  • Primary screen against recombinant nadA (10,000-100,000 compounds)

  • Secondary confirmation assays with dose-response curves

  • Counter-screening against related enzymes to assess selectivity

  • Whole-cell assays against Xylella fastidiosa cultures

• Hit characterization:

  • Mechanism of inhibition studies (competitive, non-competitive, uncompetitive)

  • Binding studies (thermal shift assays, surface plasmon resonance)

  • Structural studies (X-ray crystallography, molecular modeling)

  • Structure-activity relationship development

• Lead optimization:

  • Medicinal chemistry to improve potency and selectivity

  • ADME properties assessment for potential in planta applications

  • Formulation development for delivery to plant xylem

This approach has been successfully applied to other NAD biosynthesis enzymes and could yield selective inhibitors of Xylella fastidiosa nadA that disrupt bacterial metabolism while having minimal impact on plant processes, potentially leading to new strategies for controlling Xylella-associated plant diseases.

What are the challenges and solutions for studying nadA in the context of mixed microbial communities in plant xylem?

Studying nadA in the context of mixed microbial communities in plant xylem presents unique challenges that require specialized approaches:

• Challenges in microbial community studies:

• Methodological approaches:

  • Metatranscriptomics: Captures gene expression from all community members, requiring bioinformatic separation of Xylella sequences

  • Selective enrichment: Culture conditions favoring Xylella fastidiosa growth

  • Species-specific molecular probes: Custom primers/probes for nadA detection

  • Single-cell techniques: Fluorescence in situ hybridization (FISH) combined with flow cytometry

  • Stable isotope probing: Tracking isotope incorporation into specific metabolic pathways

• Experimental design for community studies:

  • Control communities with defined compositions

  • Comparison of healthy vs. infected plants

  • Monitoring community changes following specific treatments

  • Integration of metadata (plant physiology, environmental conditions)

• Data integration approaches:

  • Correlation networks linking nadA expression with community composition

  • Metabolomic profiling to identify community-level metabolic interactions

  • Modeling approaches to predict community dynamics

By combining these approaches, researchers can gain insights into how nadA expression and activity in Xylella fastidiosa are influenced by the surrounding microbial community, and conversely, how nadA-dependent metabolism affects community structure and function within the plant xylem environment .

How can structural comparisons between Xylella fastidiosa nadA and human NAD biosynthetic enzymes inform selective inhibitor design?

Structural comparisons between Xylella fastidiosa nadA and human NAD biosynthetic enzymes provide crucial insights for designing selective inhibitors that target bacterial metabolism while minimizing effects on host systems:

• Key structural differences for selective targeting:

• Structural biology approaches:

  • X-ray crystallography of recombinant nadA with various ligands

  • Homology modeling based on related bacterial structures

  • Molecular dynamics simulations to identify transient pockets

  • Comparison with human quinolinate phosphoribosyltransferase

• Rational inhibitor design strategy:

  • Fragment-based screening targeting bacterial-specific pockets

  • Structure-based virtual screening against bacterial nadA models

  • Focus on inhibitor properties suitable for plant vascular system delivery

  • Design of covalent inhibitors targeting bacterial-specific residues

• Selectivity assessment:

  • Counter-screening against human NAD biosynthetic enzymes

  • Cellular toxicity testing in plant and human cell models

  • Computational prediction of off-target interactions

By exploiting the fundamental differences in NAD biosynthesis between bacteria and eukaryotes (aspartate pathway vs. kynurenine pathway) , researchers can design inhibitors that selectively target Xylella fastidiosa metabolism. This approach leverages the evolutionary divergence in these pathways to develop compounds with a favorable therapeutic index for controlling Xylella infections in plants while minimizing environmental impact.

What considerations are important when translating in vitro findings about nadA inhibitors to in planta control strategies?

Translating in vitro findings about nadA inhibitors to effective in planta control strategies for Xylella fastidiosa requires addressing several critical considerations:

• Pharmacokinetic challenges in plant systems:

  • Uptake and translocation: Inhibitors must be formulated to move through plant vascular systems

  • Stability in xylem environment: Compounds must resist degradation by plant enzymes and microbial communities

  • Distribution within plants: Achieving effective concentrations throughout infected regions

  • Persistence: Maintaining therapeutic concentrations for extended periods

• Delivery methods optimization:

  • Trunk injection systems similar to tetracycline delivery methods

  • Foliar applications with appropriate adjuvants for systemic translocation

  • Soil drenches for root uptake and systemic distribution

  • Nanoformulations to enhance stability and targeting

• Efficacy translation framework:

• Practical application considerations:

  • Economic feasibility for agricultural implementation

  • Integration with existing management practices

  • Environmental impact assessment

  • Regulatory requirements for agricultural chemical approval

• Monitoring methodologies:

  • RT-qPCR assays targeting cvaC-1 or other markers to assess bacterial viability and growth

  • Symptom development tracking through standardized disease indices

  • Xylem sap analysis for both inhibitor concentrations and bacterial metabolites

Successful translation requires a multidisciplinary approach combining expertise in chemistry, plant physiology, bacteriology, and agricultural practices. Pilot field trials with careful monitoring of both inhibitor distribution and bacterial responses are essential steps before widespread implementation of nadA inhibitor-based control strategies.

How might advances in structural biology accelerate the development of selective nadA inhibitors?

Advances in structural biology are poised to significantly accelerate the development of selective nadA inhibitors through several emerging technologies and approaches:

• Cryo-electron microscopy (cryo-EM) applications:

  • Determination of high-resolution structures without crystallization

  • Visualization of nadA in different conformational states

  • Structural characterization of nadA within larger protein complexes

  • Potential for structure determination in near-native environments

• Computational advances:

  • Artificial intelligence-driven protein structure prediction (e.g., AlphaFold)

  • Molecular dynamics simulations with enhanced sampling techniques

  • Quantum mechanics/molecular mechanics (QM/MM) approaches for reaction mechanism elucidation

  • Virtual screening of ultra-large compound libraries (>1 billion compounds)

• Fragment-based drug discovery integration:

  • NMR-based fragment screening for weak but efficient binders

  • X-ray crystallography screening of fragment libraries

  • Computational fragment growing and merging strategies

  • Development of fragment libraries targeting [Fe-S] cluster-containing enzymes

• Emerging structural approaches:

  • Room-temperature crystallography to capture physiologically relevant states

  • Time-resolved crystallography to visualize catalytic intermediates

  • Neutron diffraction for precise hydrogen atom positioning

  • Serial femtosecond crystallography at X-ray free electron lasers

These technologies will provide unprecedented insights into nadA structure, dynamics, and catalytic mechanism, enabling the design of inhibitors that exploit unique features of the bacterial enzyme while avoiding cross-reactivity with host proteins. Particularly promising is the potential to identify allosteric sites and transient pockets that may not be evident in static crystal structures but could provide highly selective targeting opportunities.

What potential exists for using nadA inhibitors in combination with other antimicrobial strategies?

The potential for using nadA inhibitors in combination with other antimicrobial strategies represents a promising approach to Xylella fastidiosa control with several advantages:

• Synergistic combination possibilities:

• Resistance management benefits:

  • Multiple targets require simultaneous mutations for resistance

  • Lower individual compound concentrations reduce selection pressure

  • Different pharmacokinetic properties may create temporal coverage gaps

• Delivery system innovations:

  • Co-formulation of compatible antimicrobials

  • Sequential application strategies based on disease stage

  • Nanoparticle encapsulation for controlled release of multiple agents

  • Exploitation of trunk injection systems demonstrated effective for tetracyclines

• Experimental approaches to evaluate combinations:

  • Checkerboard assays to identify synergistic, additive, or antagonistic interactions

  • Time-kill studies to characterize temporal aspects of combination effects

  • In planta trials with multiple application strategies

  • Long-term resistance development monitoring

• Additional combination partners:

  • RNA-based technologies (RNAi, antisense)

  • CRISPR-Cas systems delivered via vectors

  • Endophytic microorganisms with antagonistic activities

  • Plant-derived antimicrobial compounds

Combination approaches involving nadA inhibitors could address many of the limitations of single-agent strategies, potentially leading to more sustainable and effective management of Xylella fastidiosa infections while reducing the environmental impact compared to broad-spectrum antimicrobials.