Recombinant Xylella fastidiosa D-alanine--D-alanine ligase (ddl)

What is D-alanine--D-alanine ligase (ddl) and what is its function in Xylella fastidiosa?

D-alanine--D-alanine ligase (Ddl) is an essential enzyme that catalyzes the ATP-dependent formation of the d-alanyl-d-alanine dipeptide, which is critical for bacterial cell wall biosynthesis . In Xylella fastidiosa, Ddl plays a fundamental role in cell wall formation, making it essential for bacterial survival . X. fastidiosa is a gram-negative bacterium that causes economically important diseases in crops including grapes, almonds, citrus, and olives . The enzyme belongs to the ATP-grasp superfamily, characterized by an atypical nucleotide-binding site known as the ATP-grasp fold .

What is the structure and composition of Xylella fastidiosa Ddl?

The Ddl enzyme in X. fastidiosa functions as a dimer. Each monomer consists of three distinct domains:

• N-terminal domain (positions approximately Met 1–Gly 104)

• Central domain (positions approximately Ala 105–Leu 192)

• C-terminal domain (positions approximately Ser 193–Thr 304)

Each subunit possesses:

• One ATP-binding site formed by the ATP-grasp fold

• Two d-Ala-binding sites positioned adjacently at the center of the monomer

The specific characteristics of X. fastidiosa Ddl include:

• Length: 304 amino acids

• Molecular mass: 32.9 kDa

• Sequence variation between strains (e.g., Temecula1/ATCC 700964 vs. 9a5c)

How does Ddl activity impact vancomycin resistance in bacteria?

The peptidoglycan composition dictated by Ddl activity directly influences vancomycin resistance in bacteria. Vancomycin binds with high affinity to peptidoglycan terminating in d-alanyl-d-alanine (d-Ala-d-Ala), resulting in vancomycin sensitivity. Conversely, vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate (d-Ala-d-Lac), conferring vancomycin resistance .

A single amino acid in the Ddl active site determines whether the enzyme functions as a dipeptide ligase or a depsipeptide ligase:

• Tyrosine (Y) → Dipeptide ligase activity → d-Ala-d-Ala production → Vancomycin sensitivity

• Phenylalanine (F) → Depsipeptide ligase activity → d-Ala-d-Lac production → Vancomycin resistance

This structure-function relationship has significant implications for both antibiotic resistance mechanisms and potential biotechnological applications.

What methodological approaches are most effective for expressing recombinant X. fastidiosa Ddl?

For successful recombinant expression of X. fastidiosa Ddl, researchers should consider the following methodological approach:

• Expression system selection: E. coli BL21(DE3) or derivatives are commonly used for Ddl expression due to their reduced protease activity and high expression yields. For more complex studies requiring proper folding, consider Lactobacillus-based expression systems that provide a gram-positive cellular environment .

• Vector design: Incorporate the following elements:

  • Strong, inducible promoter (T7 or pSIP)

  • Appropriate fusion tags (His6 or GST) for purification

  • Recognition sites for precise tag removal

  • Codon optimization for the host expression system

• Expression conditions optimization:

  • Induction parameters: IPTG concentration (0.1-1.0 mM) or induction peptide (1-8 ng/ml for pSIP system)

  • Temperature (typically 16-25°C for improved solubility)

  • Duration (4-24 hours depending on temperature)

  • Media composition (enriched media like LB or minimal media for isotope labeling)

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Buffer optimization: Include potassium ions (10-50 mM) to maintain enzyme stability

When expressing mutant variants, careful consideration of amino acid substitutions is critical, especially those affecting the active site (e.g., F260Y in L. plantarum or F258Y in L. reuteri) that can significantly alter enzyme activity and bacterial phenotype .

How can researchers effectively measure and characterize the kinetic properties of recombinant X. fastidiosa Ddl?

Characterization of X. fastidiosa Ddl kinetic properties requires multiple complementary approaches:

• ATP-Dependent Activity Assay:

  • Principle: Measures ADP formation using coupled enzyme assays (pyruvate kinase and lactate dehydrogenase)

  • Detection: NADH oxidation measured at 340 nm

  • Components: ATP (1-10 mM), d-Ala (0.1-10 mM), MgCl₂ (10 mM), potassium ions (50 mM), purified Ddl (50-200 nM)

• Dipeptide Formation Assay:

  • HPLC analysis of d-Ala-d-Ala formation

  • LC-MS/MS for precise quantification

  • Vancomycin-based fluorescence polarization assay

• Monovalent Cation Activation Analysis:

  • Compare enzyme activity with different monovalent cations (K⁺, Rb⁺, NH₄⁺, Cs⁺, Na⁺, Li⁺)

  • Determine activation order (typically K⁺≈ Rb⁺> NH₄⁺≈ Cs⁺> Na⁺≈ Li⁺)

• Key Kinetic Parameters to Determine:

For accurate comparison across experimental conditions, standardize buffer composition, temperature (typically 25°C or 37°C), and pH (typically 7.5-8.0) .

What is the role of Ddl in X. fastidiosa pathogenicity and how might it be exploited as a target for disease control?

The relationship between Ddl and X. fastidiosa pathogenicity is multifaceted:

• Cell Wall Integrity and Stress Response:

  • Ddl is essential for peptidoglycan synthesis, which maintains cell wall integrity during plant colonization

  • X. fastidiosa relies on proper cell wall structure when transitioning between plant hosts and insect vectors

  • The Rpf cell-cell communication system, which regulates virulence genes, interacts with cell wall components dependent on Ddl activity

• Biofilm Formation and Movement:

  • Cell wall modifications affect attachment to xylem vessels and insect vectors

  • Ddl activity influences the expression of genes involved in biofilm formation (e.g., hxfA and hxfB)

  • Quorum sensing systems that regulate virulence via the diffusible signal factor (DSF) have demonstrated connections to cell wall metabolism and integrity

• Potential Intervention Strategies:

  • Enzyme inhibitors specifically targeting X. fastidiosa Ddl

  • Disruption of potassium-dependent activation pathways

  • Engineering bacteriophages to deliver modified Ddl genes that increase antibiotic susceptibility

  • Expression of recombinant Ddl variants with dominant-negative effects on native enzyme function

Experimental evidence demonstrates that disrupting X. fastidiosa cell wall biosynthesis can significantly impact its ability to colonize plants and be transmitted by insect vectors, suggesting Ddl as a promising target for disease management strategies .

How can researchers design experiments to investigate the impact of site-directed mutations on X. fastidiosa Ddl function?

A comprehensive experimental design for investigating site-directed mutations in X. fastidiosa Ddl should include:

• Mutation Selection Strategy:

  • Active site residues: Focus on the glutamate residues equivalent to those shown to be critical in related species (e.g., E282 in some species)

  • Substrate binding pocket: Target residues that coordinate ATP or d-Ala binding

  • Monovalent cation binding site: Residues involved in K⁺ coordination (typically E87, M114, K116, E282, N284)

  • Dimer interface: Mutations affecting oligomerization

• Mutagenesis Protocol:

  • Use overlap extension PCR or Q5 site-directed mutagenesis for precise nucleotide changes

  • Design primers with 15-20 bp flanking sequences on each side of the mutation

  • Include silent mutations to introduce restriction sites for screening

  • Verify mutations by DNA sequencing before expression

• Functional Analysis Pipeline:

• In vivo Validation:

  • Generate X. fastidiosa strains with chromosomal mutations in ddl

  • Assess changes in vancomycin susceptibility, as demonstrated with F258Y mutations in related bacteria

  • Evaluate alterations in biofilm formation, motility, and virulence in plant models

  • Measure changes in cell wall composition using mass spectrometry

This approach has been successfully employed in related systems, as demonstrated by studies where two substitutions of glutamate to alanine in the Ddl active site rendered the enzyme incapable of producing its normal product while maintaining other functions .

What are the key considerations for using X. fastidiosa Ddl as a counterselection marker in genetic experiments?

Using X. fastidiosa Ddl as a counterselection marker requires careful consideration of the following factors:

• Mechanism of Counterselection:

  • Expression of dipeptide ligase Ddl in vancomycin-resistant bacteria increases their sensitivity to vancomycin in a dose-dependent manner

  • The effectiveness depends on the peptidoglycan composition of the target bacterium

  • A specific amino acid substitution (F258Y in L. reuteri or equivalent position in X. fastidiosa) converts the enzyme to a dipeptide ligase, increasing vancomycin sensitivity

• Vector Design Requirements:

  • Suicide vector containing the modified ddl gene

  • Appropriate promoter for expression in the target bacterium

  • Incorporation of homologous flanking regions for targeted integration

  • Careful control of expression levels using titratable promoters

• Protocol Optimization:

  • Vancomycin concentration must be empirically determined for each bacterial species

  • Induction levels for Ddl expression should be titrated (1-8 ng/ml of induction peptide)

  • Incubation time and temperature affect selection efficiency

  • Verification of recombination events by PCR analysis is essential

• Experimental Data From Related Systems:

• Verification Methods:

  • PCR screening to confirm the desired recombinant genotype

  • Sequence analysis of integration sites

  • Phenotypic testing for vancomycin sensitivity

  • Quantitative RT-PCR to measure the relative transcript levels of recombinant vs. native ddl

This counterselection approach has been successfully demonstrated in lactobacilli, where researchers achieved markerless deletions with selection efficiencies allowing identification of recombinant genotypes in approximately half the time required by conventional approaches .

How can researchers resolve contradictory data when studying the structure-function relationship of X. fastidiosa Ddl?

When confronted with contradictory data in X. fastidiosa Ddl research, implement this systematic approach:

• Identify Sources of Experimental Variability:

  • Strain differences: Compare sequences of Ddl from various X. fastidiosa strains (e.g., Temecula1/ATCC 700964 vs. 9a5c)

  • Expression systems: Results may differ between E. coli and native or related hosts

  • Assay conditions: Particularly potassium concentration, which significantly affects Ddl activity

  • Protein purification methods: Tag interference or improper folding

  • Experimental design: Sample size, statistical power, and appropriate controls

• Cross-Validation Strategy:

• Computational Approaches:

  • Molecular dynamics simulations to evaluate conformational changes

  • Quantum mechanics/molecular mechanics (QM/MM) calculations for reaction mechanisms

  • Structural comparisons with homologous enzymes from related bacteria

  • Phylogenetic analysis to identify evolutionary patterns in enzyme function

• Integrated Data Analysis:

  • Implement Bayesian statistical approaches for hypothesis testing

  • Use machine learning to identify patterns in complex datasets

  • Develop mechanistic models that incorporate all experimental variables

  • Consider alternative hypotheses that could reconcile seemingly contradictory results

• Case Study Example:
  In studies of toxin-antitoxin systems in X. fastidiosa, researchers encountered contradictory results regarding translation regulation mechanisms. Further investigation revealed that "toxin expression may not be solely responsible for translation regulation," indicating more complex regulatory networks than initially hypothesized . Similar complex regulation may occur with Ddl, requiring systematic investigation of multiple factors simultaneously.

What are the latest methodological advances for studying Ddl interactions with other proteins in the X. fastidiosa cell wall synthesis pathway?

Contemporary methodologies for investigating Ddl protein interactions in X. fastidiosa include:

• Proximity-Dependent Biotinylation (BioID/TurboID):

  • Fusion of Ddl with a biotin ligase (BioID2 or TurboID)

  • Expression in X. fastidiosa or surrogate hosts

  • Biotinylation of proximal proteins followed by streptavidin pulldown

  • Mass spectrometry identification of interaction partners

  • Advantages: Captures transient interactions and works in native cellular contexts

• Chemical Crosslinking Mass Spectrometry (XL-MS):

  • Treatment of purified Ddl or cell lysates with crosslinkers (e.g., DSS, BS3)

  • Digestion and enrichment of crosslinked peptides

  • High-resolution MS/MS analysis

  • Computational modeling of protein interaction interfaces

  • Advantages: Provides spatial constraints for structural modeling of complexes

• Cryo-Electron Microscopy of Multi-Protein Complexes:

  • Isolation of native cell wall synthesis machinery

  • Vitrification and high-resolution imaging

  • 3D reconstruction of protein complexes

  • Fitting of known crystal structures into electron density maps

  • Advantages: Visualizes large assemblies in near-native states

• CRISPR Interference (CRISPRi) Interaction Mapping:

  • Systematic CRISPRi knockdown of genes in X. fastidiosa

  • Measurement of epistatic interactions with ddl mutations

  • Construction of genetic interaction networks

  • Identification of functional relationships between proteins

  • Advantages: Reveals functional dependencies not detectable by physical interaction studies

• Integrative Structural Biology Workflow:

When studying Ddl interactions, researchers should consider its relationship with the Rpf cell-cell communication system in X. fastidiosa, which coordinates the expression of virulence genes through diffusible signal factors and may functionally interact with cell wall synthesis pathways .

What emerging technologies show promise for targeting X. fastidiosa Ddl as a strategy for controlling plant diseases?

Emerging technologies targeting X. fastidiosa Ddl show significant promise for plant disease control:

• CRISPR-Cas Antimicrobials:

  • Phage-delivered CRISPR-Cas systems targeting ddl

  • Sequence-specific antimicrobials that avoid disrupting beneficial microbiota

  • Delivery via engineered bacteriophages or nanoparticles

  • Challenge: Developing effective delivery systems to reach X. fastidiosa in plant xylem

• Peptide-Based Ddl Inhibitors:

  • Rational design based on transition-state analogs

  • Peptidomimetics that compete with d-Ala binding

  • Enhanced stability and plant vascular mobility

  • Example approach: D-cycloserine derivatives with improved specificity for X. fastidiosa Ddl

• RNA-Based Technologies:

  • Antisense oligonucleotides targeting ddl mRNA

  • Small interfering RNAs delivered via engineered plant viruses

  • Host-induced gene silencing through transgenic plants expressing dsRNA

  • Advantage: Can be expressed by the plant for continuous protection

• Structural Vaccinology Approach:

  • Identification of surface-exposed epitopes unique to X. fastidiosa Ddl

  • Development of antibodies or nanobodies targeting these regions

  • Engineering beneficial endophytes to secrete these inhibitory proteins

  • Potential for long-lasting protective colonization of plant tissues

• Combination Strategies With Experimental Data:

These approaches build upon understanding how mutations in X. fastidiosa RpfF and related proteins affect DSF production and bacterial virulence, suggesting combinatorial strategies targeting both signaling and cell wall synthesis could be particularly effective .

How might comprehensive knowledge of X. fastidiosa Ddl structure and function contribute to broader understanding of bacterial adaptation and evolution?

Comprehensive investigation of X. fastidiosa Ddl provides valuable insights into bacterial adaptation and evolution:

• Evolutionary Trajectory of Substrate Specificity:

  • Comparative analysis of Ddl across Xylella and Xanthomonas species reveals evolutionary patterns

  • Single amino acid changes (F/Y toggle) in the active site determine substrate specificity and vancomycin resistance

  • This represents a clear example of how minimal genetic changes can drive significant phenotypic adaptations

  • Phylogenetic analysis of 173 Lactobacillus species showed that 140 are predicted to be vancomycin-resistant based on Ddl sequence, demonstrating the evolutionary conservation of this mechanism

• Host-Pathogen Co-evolution:

  • X. fastidiosa strains show host-specific adaptations affecting virulence genes and cell wall composition

  • Experimental testing of X. fastidiosa subspecies multiplex on 17 plant species revealed host-specific infection patterns

  • Cell wall modifications influenced by Ddl activity may contribute to evasion of plant immune responses

  • Understanding this co-evolutionary process provides insights into bacterial adaptation to different plant hosts

• Horizontal Gene Transfer and Functional Integration:

  • Analysis of genomic contexts surrounding ddl genes can identify horizontal gene transfer events

  • Integration of acquired genes into existing metabolic and regulatory networks

  • Evidence from toxin-antitoxin systems in X. fastidiosa suggests complex integration of horizontally acquired elements

  • Studying these processes enhances understanding of bacterial genome plasticity and adaptation

• Stress Response and Persistence Mechanisms:

  • Ddl activity connects to broader stress response networks

  • Cell wall modifications contribute to survival under environmental stresses

  • These connections illuminate how bacteria balance growth requirements with stress resistance

  • The relationship between Ddl and diffusible signal factors reveals sophisticated regulatory networks

• Evolutionary Framework for Antibiotic Resistance:

  • The natural variation in Ddl specificity provides a model for understanding the evolution of antibiotic resistance

  • Insights from X. fastidiosa can inform broader questions about the emergence of resistance in clinical pathogens

  • The balance between antivirulence effects and transmission efficiency demonstrates evolutionary trade-offs in bacterial pathogens

This research exemplifies how detailed molecular understanding of a single enzyme can illuminate broader evolutionary principles operating across diverse bacterial taxa.