Recombinant Xylella fastidiosa Dihydrodipicolinate synthase (dapA)

Basic Research Questions

• What is dihydrodipicolinate synthase (dapA) and what is its function in Xylella fastidiosa?

  Dihydrodipicolinate synthase (DHDPS, encoded by the dapA gene) is a key enzyme in the lysine biosynthesis pathway via the diaminopimelate route in prokaryotes, including Xylella fastidiosa. The enzyme catalyzes the condensation of L-aspartate-β-semialdehyde and pyruvate to form 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate through a ping-pong mechanism, where pyruvate binds to the enzyme by forming a Schiff base with a lysine residue .

  In X. fastidiosa, this enzyme is part of an extensive biosynthetic capability that is essential for a xylem-dwelling bacterium. While most genes found in E. coli necessary for amino acid synthesis are identified in X. fastidiosa, some genes are bi-functional, including a notable diaminopimelate decarboxylase/aspartate kinase (XF1116) that catalyzes both the first and last steps of lysine biosynthesis . This adaptation is significant as it may represent an evolutionary optimization of the lysine biosynthesis pathway in X. fastidiosa.

• What molecular techniques are most effective for cloning the dapA gene from Xylella fastidiosa?

  For effective cloning of the dapA gene from X. fastidiosa, researchers should consider:

  • Primer design: Using genome sequences available from multiple X. fastidiosa strains to design primers with appropriate restriction sites for downstream cloning.

  • PCR optimization: Given the high GC content and repetitive regions in X. fastidiosa, PCR conditions often require optimization with:

    • Higher denaturation temperatures (95-98°C)

    • DMSO or other PCR enhancers (5-10%)

    • Specialized polymerases for high-fidelity amplification

  A recommended protocol includes:

  1. Genomic DNA extraction using specialized kits for gram-negative bacteria

  2. PCR amplification with optimized conditions

  3. Cloning into an intermediate vector (e.g., pGEM-T Easy)

  4. Sequence verification before subcloning into expression vectors

  For recombinant expression, vectors containing the PemI/PemK plasmid addiction system have shown to provide stable maintenance in X. fastidiosa without antibiotic selection, which is particularly useful for complementation studies both in vitro and in planta .

• How can researchers verify the identity and functionality of recombinant X. fastidiosa dapA?

  Verification of recombinant X. fastidiosa dapA should employ multiple approaches:

  • Sequence verification: Complete sequencing of the cloned gene to confirm the absence of mutations.

  • Expression analysis: Western blotting using specific antibodies against the target protein or epitope tags (His, FLAG, etc.).

  • Enzymatic activity assay: Measure DHDPS activity through:

    • Spectrophotometric detection of product formation at 270 nm

    • Coupled enzyme assays measuring consumption of NADPH

    • Detection of the imino intermediate using o-aminobenzaldehyde, which yields a dihydroquinazolium compound with absorbance at 440 nm

  • Complementation studies: Testing the ability of the recombinant dapA to rescue growth in E. coli dapA-deficient strains in the absence of lysine supplementation.

  The enzymatic activity should be measured under standardized conditions (pH 8.0, 30°C) to allow comparison with DHDPS from other bacterial species.

• What are the key differences between X. fastidiosa dapA and related enzymes in other bacterial species?

  X. fastidiosa dapA shares structural and catalytic similarities with DHDPS enzymes from other bacterial species, but with notable differences:

  Feature	X. fastidiosa DHDPS	E. coli DHDPS	Mycobacterium tuberculosis DHDPS
  Quaternary structure	Tetrameric	Tetrameric	Tetrameric
  Lysine inhibition	Moderate	Strong	Weak
  Catalytic efficiency (kcat/Km for pyruvate)	Intermediate	High	Low
  Optimal pH	7.5-8.0	8.0-8.5	7.0-7.5
  Thermal stability	Moderate	Moderate	High

  The X. fastidiosa DHDPS likely represents an adaptation to the unique environmental conditions of the xylem vessels, where nutrient availability fluctuates and where the enzyme must function optimally at the physiological pH of xylem sap (typically pH 6.5-7.5).

• What expression systems are most suitable for producing recombinant X. fastidiosa dapA?

  Several expression systems have been employed for recombinant X. fastidiosa proteins, with varying success for dapA:

  • E. coli systems: BL21(DE3) strains with pET vectors typically yield high expression levels, though optimization of induction conditions is often necessary to prevent inclusion body formation.

  • X. fastidiosa-specific vectors: Plasmids utilizing native X. fastidiosa replication origins along with high-copy-number pUC origins for propagation in E. coli cloning strains, such as pXf20pemIK, can be used for complementation studies .

  • Broad-host-range vectors: Medium to low-copy-number plasmids based on the pBBR1 backbone (e.g., pBBR5pemIK) are maintained for extended periods without antibiotic selection .

  For structural studies requiring high protein yields, the E. coli system with codon optimization and fusion tags (particularly His6 or MBP) typically provides the best results, while complementation studies benefit from X. fastidiosa-compatible vectors.

Advanced Research Questions

• How does homologous recombination affect dapA gene variation across different X. fastidiosa subspecies?

  Homologous recombination plays a significant role in X. fastidiosa genome evolution and potentially in dapA gene variation. Research indicates:

  • X. fastidiosa subspecies show varying levels of recombination, with X. fastidiosa subsp. fastidiosa showing lower recombination rates (averaging 3.22 of 622 core genes) compared to X. fastidiosa subsp. multiplex (averaging 9.60 recombining genes) .

  • Intersubspecific homologous recombination (IHR) can reach up to 15.30% in the core genome for some subspecies like X. fastidiosa subsp. morus .

  • The relative effect of recombination to mutation (r/m) has been calculated at 2.259 across X. fastidiosa , indicating recombination has a greater impact on genetic diversity than point mutations.

  An introgression test can detect recombination in X. fastidiosa better than standard recombination tests. This test has identified recombination regions totaling 6,053 bp in six loci, while the best-performing standard tests only identified 30% of these regions .

  For dapA specifically, researchers should examine:

  1. Sequence variations in dapA genes across subspecies

  2. Potential recombination breakpoints within or flanking the dapA gene

  3. The impact of recombination events on dapA gene expression and enzyme function

• What methodologies are most effective for studying the kinetics of X. fastidiosa dapA with various substrates and inhibitors?

  For rigorous kinetic studies of X. fastidiosa dapA, researchers should implement:

  • Steady-state kinetics:

    • Continuous spectrophotometric assays measuring pyruvate consumption or product formation

    • Initial velocity measurements across a range of substrate concentrations (typically 0.05-10 × Km)

    • Data fitting to appropriate models (Michaelis-Menten, substrate inhibition, allosteric)

  • Pre-steady-state kinetics:

    • Stopped-flow spectroscopy to capture rapid formation of enzyme-substrate complexes

    • Rapid quench-flow techniques to analyze reaction intermediates

  • Inhibition studies:

    • Determination of inhibition constants (Ki) for competitive, uncompetitive, and non-competitive inhibitors

    • Analysis of inhibition mechanisms through Dixon and Cornish-Bowden plots

  A standard protocol for kinetic analysis includes:

  1. Purifying recombinant enzyme to >95% homogeneity

  2. Determining enzyme concentration through absorbance at 280 nm

  3. Establishing optimal reaction conditions (pH, temperature, buffer composition)

  4. Measuring initial velocities under conditions where <10% of substrate is consumed

  5. Data analysis using nonlinear regression software

  For accurate kinetic parameters, researchers should ensure that:

  • The assay is linear with respect to time and enzyme concentration

  • The assay conditions mimic physiological conditions when possible

  • Multiple batches of enzyme are tested to ensure reproducibility

• How can site-directed mutagenesis be used to study the catalytic mechanism of X. fastidiosa dapA?

  Site-directed mutagenesis provides valuable insights into the catalytic mechanism of X. fastidiosa dapA through systematic modification of key residues. An effective approach includes:

  1. Target residue identification:

     • Conserved active site residues identified through sequence alignment with characterized DHDPS enzymes

     • Residues implicated in substrate binding, catalysis, or allosteric regulation

     • Residues unique to X. fastidiosa DHDPS that may contribute to its specific properties

  2. Mutagenesis strategy:

     • Conservative substitutions to probe electrostatic and steric roles (e.g., Lys → Arg, Asp → Glu)

     • Elimination of functional groups (e.g., Lys → Ala, Tyr → Phe)

     • Introduction of non-canonical amino acids for mechanistic studies

  3. Functional characterization of mutants:

     • Kinetic parameter determination (kcat, Km, kcat/Km)

     • pH-rate profiles to identify ionizable groups

     • Thermal stability analysis through differential scanning fluorimetry

     • Structural analysis through circular dichroism or crystallography

  A comprehensive mutagenesis study should systematically characterize:

  Residue Type	Example Residues	Mutations	Expected Effect
  Schiff base formation	Conserved lysine	K→A, K→R	Loss of activity
  Substrate binding	Conserved arginines	R→K, R→A	Increased Km
  Catalytic residues	Conserved tyrosine	Y→F, Y→A	Decreased kcat
  Allosteric site	Residues in C-terminal domain	Multiple	Altered regulation

  This systematic approach can elucidate the roles of specific residues in catalysis and provide insights into potential differences between X. fastidiosa DHDPS and related enzymes.

• What are the challenges in crystallizing recombinant X. fastidiosa dapA for structural studies?

  Crystallizing recombinant X. fastidiosa dapA presents several challenges requiring systematic approaches:

  1. Protein heterogeneity issues:

     • Optimize expression to minimize proteolysis by including protease inhibitors

     • Employ size-exclusion chromatography as a final purification step

     • Verify homogeneity through dynamic light scattering (target polydispersity <15%)

     • Consider limited proteolysis to remove disordered regions that may impede crystallization

  2. Crystallization condition optimization:

     • Employ sparse matrix screens followed by systematic optimization

     • Test protein at multiple concentrations (typically 5-20 mg/ml)

     • Include substrate analogs or inhibitors to stabilize active site conformation

     • Test crystallization with and without lysine (allosteric regulator)

  3. Crystal quality improvement:

     • Implement seeding techniques (micro- and macroseeding)

     • Use additives screening to identify crystal-improving compounds

     • Consider surface entropy reduction through mutagenesis

     • Explore crystallization at different temperatures (4°C, 18°C, 25°C)

  4. X-ray diffraction challenges:

     • Test cryoprotectant conditions systematically

     • Consider room-temperature data collection for radiation-sensitive crystals

     • Explore heavy atom derivatives for phasing if molecular replacement fails

  Researchers have found success with bacterial DHDPS crystallization using:

  • Hanging drop vapor diffusion with 15-25% PEG 3350/4000/8000

  • pH range 6.5-8.5 in various buffers (HEPES, Tris, phosphate)

  • Addition of divalent cations (Mg²⁺, Ca²⁺) at 5-10 mM

  • Crystallization in the presence of pyruvate (5-10 mM)

• How can comparative genomics approaches be used to study the evolution of dapA in X. fastidiosa?

  Comparative genomics provides powerful insights into dapA evolution in X. fastidiosa. A comprehensive approach should include:

  1. Phylogenetic analysis:

     • Multiple sequence alignment of dapA genes from diverse X. fastidiosa strains and related species

     • Construction of maximum likelihood trees using appropriate substitution models

     • Reconciliation of gene trees with species trees to identify potential horizontal gene transfer events

  2. Selection analysis:

     • Calculation of dN/dS ratios across the dapA coding sequence

     • Branch-site tests to identify episodic selection on specific lineages

     • Sliding window analysis to identify regions under selection

  3. Recombination detection:

     • Application of multiple recombination detection methods (RDP, GeneConv, MaxChi, Chimaera, 3Seq)

     • Implementation of introgression tests which have proven more effective in X. fastidiosa

     • Characterization of recombination breakpoints and their conservation across strains

  4. Structural consequences assessment:

     • Mapping of variable residues onto structural models

     • Prediction of functional consequences of observed variations

     • Correlation of sequence variations with ecological niches or host specificities

  Research has shown that X. fastidiosa subspecies are under different selective pressures, with limited overlap in genes showing high dN/dS values . For dapA specifically, researchers should examine whether it falls into the core or accessory genome categories, as these show different patterns of recombination and selection across subspecies.

• What experimental approaches can effectively determine the role of dapA in X. fastidiosa virulence and host adaptation?

  To determine the role of dapA in X. fastidiosa virulence and host adaptation, researchers should implement:

  1. Gene knockout and complementation studies:

     • Generate dapA deletion mutants using allelic exchange

     • Create complemented strains using stable vectors like pXf20pemIK or pBBR5pemIK that maintain without antibiotic selection for up to 14 weeks in grapevines

     • Include appropriate controls (wild-type, vector-only)

  2. Virulence assessment in planta:

     • Inoculate host plants using standardized methods

     • Monitor disease progression through symptom severity scales

     • Quantify bacterial populations in planta at different time points

     • Analyze xylem vessel occlusion patterns, which are hallmarks of X. fastidiosa infection

  3. Metabolomic analyses:

     • Compare lysine and other amino acid levels in wild-type and mutant strains

     • Analyze metabolite profiles in infected vs. healthy plant tissues

     • Investigate potential lysine-derived signaling molecules

  4. Transcriptomic analyses:

     • Perform RNA-seq on wild-type and dapA mutants under relevant conditions

     • Identify genes co-regulated with dapA or affected by dapA mutation

     • Compare transcriptional responses in different host plant species

  5. Biofilm formation assessment:

     • Quantify attachment, aggregation, and mature biofilm formation

     • Visualize biofilm structure through confocal microscopy

     • Correlate biofilm phenotypes with extracellular protein profiles

  Research has shown that X. fastidiosa virulence involves biofilm formation, cell-cell signaling (via diffusible signaling factor), and extracellular enzymes . Investigating how dapA activity relates to these virulence mechanisms would provide valuable insights into its role in pathogenicity.

• How can molecular dynamics simulations complement experimental studies of X. fastidiosa dapA?

  Molecular dynamics (MD) simulations provide valuable insights into X. fastidiosa dapA structure and function that complement experimental approaches:

  1. Structural dynamics exploration:

     • Simulate the enzyme's behavior in solution over nanosecond to microsecond timescales

     • Identify flexible regions that may be involved in substrate binding or allosteric regulation

     • Characterize conformational changes upon substrate binding or inhibitor interaction

  2. Substrate binding mechanism elucidation:

     • Calculate binding free energies using methods like MM-PBSA or FEP

     • Identify key residue interactions through interaction energy decomposition

     • Characterize the water network in the active site and its role in catalysis

  3. Allosteric regulation investigation:

     • Simulate the effects of lysine binding at allosteric sites

     • Identify communication pathways between allosteric and active sites

     • Quantify changes in protein dynamics upon allosteric effector binding

  4. Mutation effects prediction:

     • Simulate the structural and dynamic consequences of mutations

     • Predict changes in substrate binding affinity and catalytic efficiency

     • Guide experimental mutagenesis by identifying promising mutation targets

  A typical MD simulation protocol includes:

  Simulation Phase	Purpose	Typical Duration
  Minimization	Remove steric clashes	N/A (energy-based)
  Heating	Gradually increase temperature to target	100-200 ps
  Equilibration	Stabilize pressure, density, and energy	1-10 ns
  Production	Sample conformational space	100 ns - 1 μs
  Analysis	Extract meaningful properties	N/A (post-processing)

  For best results, researchers should:

  • Use well-validated force fields appropriate for proteins (AMBER, CHARMIP, OPLS)

  • Include explicit solvent with appropriate counter-ions

  • Perform multiple replicate simulations to ensure statistical significance

  • Validate simulation predictions through experimental approaches

• What methodologies are most effective for studying the regulation of dapA expression in X. fastidiosa under different environmental conditions?

  To study dapA expression regulation in X. fastidiosa under different environmental conditions, researchers should employ:

  1. Transcriptional analysis:

     • Quantitative RT-PCR for targeted gene expression analysis

     • RNA-seq for genome-wide transcriptional profiling

     • 5′ RACE to identify transcription start sites and potential alternative promoters

     • Northern blotting to detect potential processing or degradation products

  2. Promoter analysis:

     • Reporter gene fusions (GFP, luciferase) to monitor promoter activity

     • Promoter deletion/mutation analysis to identify regulatory elements

     • Electrophoretic mobility shift assays to detect protein-DNA interactions

     • DNase I footprinting to precisely locate transcription factor binding sites

  3. Environmental condition testing:

     • Media with varying nutrient availability (particularly nitrogen sources)

     • Different pH conditions mimicking host xylem environments (pH 6.5-7.5)

     • Oxidative stress conditions (H₂O₂, paraquat)

     • Co-culture with other microorganisms found in the xylem environment

  4. In planta expression studies:

     • RT-qPCR from infected plant tissue at different stages of infection

     • In situ hybridization to localize gene expression in infected tissues

     • RNA-seq from laser-capture microdissected infected tissues

     • Comparison of expression patterns in susceptible versus resistant hosts

  A comprehensive study should examine expression under conditions relevant to X. fastidiosa's lifestyle:

  Condition Type	Specific Conditions	Rationale
  Nutrient availability	Varying nitrogen sources	Amino acid biosynthesis regulation
  Physical parameters	pH 5.5-8.0, 18-28°C	Mimics host xylem conditions
  Cell density	Early vs. late growth phase	Quorum sensing effects
  Biofilm formation	Planktonic vs. biofilm cells	Lifestyle-specific regulation
  Plant extracts	Susceptible vs. resistant hosts	Host-specific signals

  Research has shown that X. fastidiosa undergoes significant transcriptomic reprogramming during host infection, with changes preceding symptom appearance . Connecting dapA regulation to these broader transcriptional networks would provide valuable insights into its role in the infection process.

• How can metabolic flux analysis be used to study the role of dapA in X. fastidiosa metabolism?

  Metabolic flux analysis (MFA) offers powerful insights into the role of dapA in X. fastidiosa metabolism through:

  1. 13C-labeled substrate experiments:

     • Feed X. fastidiosa cultures with 13C-labeled glucose or other carbon sources

     • Analyze isotope incorporation patterns in amino acids and other metabolites

     • Determine relative flux distributions across central carbon metabolism and the lysine biosynthesis pathway

     • Compare wild-type and dapA mutant strains to identify metabolic rerouting

  2. Metabolic network reconstruction:

     • Create a genome-scale metabolic model of X. fastidiosa

     • Include reactions catalyzed by dapA and other lysine biosynthesis enzymes

     • Incorporate gene-protein-reaction associations

     • Validate the model using experimental growth data

  3. Flux balance analysis (FBA):

     • Predict optimal flux distributions under different growth conditions

     • Perform in silico gene knockout simulations to predict the metabolic impact of dapA deletion

     • Identify potential synthetic lethal interactions with dapA

     • Simulate metabolic adaptation to different host environments

  4. Integration with other -omics data:

     • Constrain flux models using transcriptomic and proteomic data

     • Correlate predicted fluxes with metabolite concentrations

     • Identify regulatory mechanisms controlling metabolic flux redistribution

  The analysis should focus on:

  Analysis Type	Key Measurements	Expected Insights
  Steady-state MFA	Isotopomer distributions	Flux partitioning at metabolic branch points
  Dynamic MFA	Time-course isotope incorporation	Temporal metabolic adaptation
  Comparative MFA	Different growth conditions	Environmental influence on metabolism
  Multi-strain MFA	Wild-type vs. mutant strains	Genetic control of metabolic flux

  For X. fastidiosa specifically, researchers should examine metabolic flux through the lysine biosynthesis pathway in relation to:

  • Growth in nutrient-limited xylem environments

  • Biofilm formation, which is critical for virulence

  • Cell wall biosynthesis, which requires diaminopimelate derived from the pathway

  • Cross-talk with other amino acid biosynthetic pathways

• What strategies can be employed to develop selective inhibitors of X. fastidiosa dapA for potential disease control?

  Developing selective inhibitors of X. fastidiosa dapA requires a multi-faceted approach:

  1. Structure-based design:

     • Utilize crystal structures or homology models of X. fastidiosa dapA

     • Identify unique features of the active site for selective targeting

     • Design inhibitors that exploit structural differences between bacterial and plant DHDPS

     • Employ molecular docking to screen virtual compound libraries

  2. High-throughput screening:

     • Develop robust activity assays suitable for automation

     • Screen diverse chemical libraries for inhibitory activity

     • Include counter-screens against plant DHDPS to ensure selectivity

     • Validate hits using orthogonal assay methods

  3. Fragment-based drug discovery:

     • Screen fragment libraries using NMR, X-ray crystallography, or surface plasmon resonance

     • Identify binding hotspots in the enzyme

     • Link or grow fragments to create more potent inhibitors

     • Optimize physicochemical properties for penetration into bacterial cells

  4. Inhibitor optimization:

     • Determine structure-activity relationships through systematic modification

     • Optimize potency, selectivity, and physicochemical properties

     • Evaluate stability in relevant biological environments

     • Assess toxicity against plant cells and beneficial microorganisms

  5. Delivery system development:

     • Design formulations for uptake by plants and translocation to xylem

     • Explore systemic acquired resistance inducers as co-treatments

     • Consider grafting applications for long-lived woody hosts

     • Develop controlled-release formulations for sustained protection

  The development pathway should include:

  Development Stage	Key Activities	Success Criteria
  Target validation	Confirm essentiality of dapA	Growth inhibition in dapA knockdown strains
  Assay development	Establish robust screening cascade	Z' factor >0.5, CV <10%
  Hit identification	Primary and confirmatory screening	IC50 <100 μM, selectivity >10-fold
  Hit-to-lead	Improve potency and properties	IC50 <1 μM, suitable physicochemical profile
  In vitro efficacy	Activity against X. fastidiosa cultures	MIC <10 μg/mL
  Ex vivo testing	Activity in infected plant tissues	Significant reduction in bacterial load
  In planta evaluation	Greenhouse and field trials	Disease severity reduction >50%

  Given the increasing importance of X. fastidiosa as a global plant pathogen affecting multiple crops , developing effective and selective inhibitors of dapA represents a promising approach for disease management.