Recombinant Vibrio vulnificus D-alanine--D-alanine ligase (ddl)

What is the function of D-alanine-D-alanine ligase in Vibrio vulnificus and why is it significant?

D-alanine-D-alanine ligase (ddl) in Vibrio vulnificus is an essential enzyme that catalyzes the ATP-dependent formation of D-alanyl-D-alanine dipeptides required for peptidoglycan biosynthesis in bacterial cell walls. The enzyme plays a critical role in maintaining cell wall integrity, which is essential for bacterial survival. Recent research has identified novel variants of these genes (named ddl6 and ddl7) located within the first gene cassette of reverse integrons in Vibrio species . This positioning is particularly significant as it represents the first reported instance of ddl genes within integrons, suggesting a unique evolutionary adaptation.

The significance of these ddl variants extends beyond their structural role in cell wall synthesis. Expression of ddl6 and ddl7 has been demonstrated to confer resistance to D-cycloserine, an antibiotic currently used for treating multidrug-resistant and extensively drug-resistant tuberculosis . This resistance mechanism highlights the clinical relevance of understanding these enzymes, as they may contribute to antibiotic treatment failures in Vibrio infections.

How do integron-located ddl genes differ from chromosomal D-alanine-D-alanine ligases?

Integron-located ddl genes in Vibrio vulnificus display several distinctive characteristics compared to conventional chromosomal ddl genes:

• Genetic context: These ddl variants (ddl6 and ddl7) are uniquely positioned as the first gene cassette within reverse integrons, whereas typical ddl genes are chromosomally encoded . This positioning likely affects expression levels and regulation.

• Phylogenetic distinctiveness: Research indicates that these integron-encoded ddl variants form a separate phylogenetic clade compared to conventional ddl enzymes, suggesting significant sequence divergence and specialized evolution .

• Functional properties: While maintaining their primary catalytic function, integron-encoded ddl variants confer resistance to D-cycloserine, possibly through structural modifications that reduce inhibitor binding while preserving enzyme activity .

• Structural similarities: Analysis of predicted 3D structures reveals that these integron-encoded Ddls show similarities to vancomycin-resistant proteins, suggesting potential evolutionary relationships or functional convergence that isn't typically observed in chromosomal ddl enzymes .

• Mutational sensitivity: Evidence suggests that a single base pair mutation in these ddl variants can significantly alter resistance levels, indicating a finely-tuned structure-function relationship that may differ from conventional ddl enzymes .

What are the most effective approaches for cloning and expressing Vibrio vulnificus ddl genes?

While the search results don't specifically detail methods for cloning ddl from Vibrio vulnificus, effective approaches can be inferred from successful cloning strategies used for other V. vulnificus genes:

When optimizing expression, consider that oxidation state affects activity—other V. vulnificus enzymes show activity in the oxidized but not the reduced form . Therefore, expression conditions should be adjusted to promote proper disulfide bond formation.

How can single-base mutations in ddl be generated and evaluated for their effect on antibiotic resistance?

Given that single base pair mutations in ddl can alter D-cycloserine resistance levels , a systematic approach to studying these mutations includes:

• Site-directed mutagenesis techniques:

  • QuikChange or Q5 site-directed mutagenesis kits for precise base substitutions

  • Overlap extension PCR for more complex modifications

  • CRISPR-Cas9 genome editing for chromosomal mutations in native context

• Target selection strategy:

  • Focus on the omega-loop region identified as potentially important for resistance properties

  • Target highly conserved residues in the active site

  • Create alanine-scanning libraries across functional domains

  • Introduce mutations that mimic natural polymorphisms

• Resistance evaluation methods:

  • Minimum inhibitory concentration (MIC) determination through broth microdilution

  • Disk diffusion assays for qualitative screening

  • Growth curve analysis in the presence of varying antibiotic concentrations

  • Time-kill assays to evaluate killing kinetics

  • Population analysis profiling to detect heteroresistance

• Functional impact assessment:

  • Enzyme activity assays comparing wild-type and mutant proteins

  • Thermal stability analysis using differential scanning fluorimetry

  • Substrate binding studies using isothermal titration calorimetry

  • Structural analysis through circular dichroism or X-ray crystallography

What biochemical assays can be used to characterize the enzymatic activity of recombinant Vibrio vulnificus ddl?

Comprehensive characterization of recombinant V. vulnificus ddl requires multiple complementary assays:

• Primary activity assays:

  • ADP release measurement: Couple ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase, monitoring absorbance decrease at 340 nm

  • Inorganic phosphate detection: Use malachite green assay to quantify released phosphate

  • Direct product detection: HPLC or LC-MS quantification of D-alanyl-D-alanine dipeptide formation

• Substrate specificity assessment:

  • Side-by-side comparison using various D-amino acids as substrates

  • Competition assays with mixed substrates to determine preference

  • Analysis of altered peptidoglycan precursors using mass spectrometry

• Inhibition studies:

  • IC50 determination for D-cycloserine and other known ddl inhibitors

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

  • Flavonoid inhibition assays to test the dietary flavonoid hypothesis

  • Recovery of activity after inhibitor removal to assess binding reversibility

• Enzyme kinetics:

  • Determination of kinetic parameters (Km, Vmax, kcat) for ATP and D-alanine

  • pH and temperature optima characterization

  • Metal ion dependency assessment

  • Effects of oxidation state on activity, given that some V. vulnificus enzymes are active only in oxidized form

• Structural stability:

  • Thermal denaturation profiles using differential scanning fluorimetry

  • Chemical denaturation studies with urea or guanidinium chloride

  • Limited proteolysis to identify stable domains and flexible regions

Why are ddl genes found within integrons in Vibrio vulnificus and what are the implications?

The presence of ddl genes within integrons represents an unusual and significant finding, as these typically encode metabolic enzymes not commonly found in mobile genetic elements. Research has uncovered several important aspects of this genetic arrangement:

• Positional significance: In Vibrio vulnificus, ddl variants (ddl6 and ddl7) are specifically located in the first position within an integron . This position is typically associated with the highest expression levels due to proximity to the integron promoter, suggesting evolutionary selection for enhanced ddl expression.

• Proposed selective pressure: A compelling hypothesis suggests that dietary flavonoids with Ddl inhibitory activity could act as environmental drivers selecting for ddl genes within integrons . This natural selection would predate clinical antibiotic use and represents a novel perspective on resistance evolution.

• Horizontal gene transfer potential: The integron location facilitates horizontal transmission of these ddl variants between bacteria, potentially accelerating the spread of resistance determinants across bacterial populations and species.

• Antibiotic resistance implications: The integron-encoded ddl variants confer resistance to D-cycloserine , suggesting that their acquisition provides a direct selective advantage in environments where this or similar compounds are present.

• Evolutionary innovation: The separate phylogenetic clade formed by these ddl variants indicates they represent a distinct evolutionary lineage , possibly adapting to specific ecological niches or selective pressures encountered by Vibrio species.

This genetic arrangement illustrates how environmental pressures can shape bacterial genome architecture and resistance mechanisms, with potential consequences for clinical antibiotic efficacy and resistance monitoring strategies.

What evidence supports the hypothesis that dietary flavonoids drive selection of ddl genes in integrons?

The hypothesis that dietary flavonoids drive the selection of ddl genes in integrons represents an innovative perspective on the evolution of antibiotic resistance determinants. While the search results don't provide extensive experimental validation, several lines of reasoning support this hypothesis:

• Known inhibitory activity: Flavonoids have documented inhibitory effects on bacterial cell wall synthesis enzymes, including potential activity against D-alanine-D-alanine ligases. This creates a natural selective pressure similar to antibiotics.

• Ecological relevance: Vibrio species inhabit aquatic environments where they encounter plant-derived compounds, including flavonoids from decomposing plant matter in estuarine ecosystems. This creates a consistent selective pressure in their natural habitat.

• Positional evidence: The location of ddl genes specifically in the first position of integrons suggests selection for maximal expression, which would be advantageous if these enzymes were targets of environmental inhibitors like flavonoids.

• Parallel to clinical resistance: The ability of these ddl variants to confer D-cycloserine resistance demonstrates their capacity to overcome enzyme inhibitors, which could have initially evolved in response to flavonoids before providing cross-resistance to clinical antibiotics.

• Evolutionary timing: The hypothesis provides a plausible explanation for the acquisition and maintenance of these genes in integrons long before the clinical use of antibiotics targeting ddl.

To fully validate this hypothesis, further research is needed, including:

• Direct demonstration of flavonoid inhibition of V. vulnificus ddl variants

• Comparative inhibition studies between integron-encoded and conventional ddl enzymes

• Experimental evolution studies exposing V. vulnificus to flavonoids to observe selection for integron-encoded ddl

How do the structural similarities between integron-encoded Ddls and vancomycin resistance proteins inform our understanding of resistance evolution?

The observed structural similarities between integron-encoded Ddls from Vibrio vulnificus and vancomycin resistance proteins provide valuable insights into convergent evolution of resistance mechanisms:

• Structural convergence: Despite potentially different evolutionary origins, these proteins appear to have converged on similar three-dimensional structures, particularly in regions critical for substrate binding and catalysis. This suggests limited structural solutions to the functional challenge of maintaining cell wall synthesis while evading inhibition.

• Resistance potential: The structural similarity supports the hypothesis that a single mutation in the omega-loop of integron-encoded Ddls could confer vancomycin resistance . This represents a concerning potential for resistance development with minimal genetic change.

• Evolutionary pathways: The structural similarities may reveal evolutionary pathways by which resistance mechanisms develop. Rather than requiring completely novel proteins, resistance may often emerge through modifications to existing enzymes that maintain their primary function while reducing inhibitor binding.

• Cross-resistance mechanisms: These structural relationships suggest potential mechanisms of cross-resistance, where adaptation to one selective pressure (such as dietary flavonoids) may inadvertently provide resistance to clinically relevant antibiotics through shared binding site modifications.

• Predictive value: Understanding these structural relationships may enable prediction of potential resistance mechanisms before they emerge clinically, allowing proactive monitoring and countermeasure development.

How do structural features of Vibrio vulnificus ddl variants contribute to D-cycloserine resistance?

The D-cycloserine resistance conferred by Vibrio vulnificus ddl variants (ddl6 and ddl7) likely stems from specific structural adaptations:

• Active site architecture: D-cycloserine typically inhibits ddl by competing with D-alanine for the substrate binding site. The integron-encoded ddl variants likely possess modified active site architectures that reduce D-cycloserine binding affinity while preserving affinity for the natural D-alanine substrate.

• Binding pocket selectivity: Small changes in binding pocket geometry can dramatically affect inhibitor recognition while maintaining substrate specificity. The separate phylogenetic clade formed by these ddl variants suggests sequence divergence that may have altered critical binding residues.

• Mutation sensitivity: The finding that a single base pair mutation can alter resistance levels indicates that specific amino acid residues play crucial roles in determining D-cycloserine interactions. These may include residues that directly contact the inhibitor or that influence the conformational dynamics of the binding site.

• Stability changes: Some resistance-conferring mutations may alter protein stability or flexibility, changing the enzyme's conformational ensemble to states less favorable for inhibitor binding while still compatible with catalysis.

• Potential allosteric effects: Mutations outside the active site may create or modify allosteric sites that regulate enzyme activity in response to inhibitor binding, potentially allowing the enzyme to adopt conformations that exclude the inhibitor.

To definitively characterize these mechanisms, crystal structures of these ddl variants in complex with substrates and inhibitors would be invaluable, complemented by mutagenesis studies targeting specific residues predicted to be involved in resistance.

What is the significance of the omega-loop in potential vancomycin resistance of integron-encoded Ddls?

The search results indicate that analysis of the predicted 3D structures of gene cassette-encoded Ddls showed similarity to vancomycin-resistant proteins, leading to the hypothesis that a single mutation in the omega-loop region could confer vancomycin resistance . This omega-loop appears to be a critical structural element with several significant implications:

• Structural determinant: In vancomycin resistance proteins like VanA and VanB, the omega-loop region helps determine the enzyme's ability to produce alternative peptidoglycan precursors ending in D-alanyl-D-lactate instead of D-alanyl-D-alanine, dramatically reducing vancomycin binding affinity.

• Mutational hotspot: The hypothesis that a single mutation in this region could confer vancomycin resistance suggests it represents a mutational hotspot where small genetic changes can have large functional consequences, representing a low genetic barrier to resistance development.

• Substrate specificity control: The omega-loop likely contributes to substrate recognition and binding, with mutations potentially altering the enzyme's ability to accommodate alternative substrates while maintaining catalytic function.

• Evolutionary conservation: The structural similarity between integron-encoded Ddls and vancomycin resistance proteins specifically in this region suggests it may be subject to convergent evolution when under similar selective pressures.

• Diagnostic and surveillance value: This region represents a potential biomarker for monitoring the emergence of vancomycin resistance, where specific mutations could serve as early warning indicators of evolving resistance.

The identification of this specific structural feature provides a focused target for further research, including site-directed mutagenesis experiments to test the hypothesis directly and structural studies to characterize the precise molecular interactions involved in potential resistance mechanisms.

How does the periplasmic environment affect the function and resistance properties of bacterial ligases like ddl?

While the search results don't directly address how the periplasmic environment affects ddl function, information about other periplasmic enzymes in Vibrio vulnificus provides relevant insights:

• Oxidation state dependency: Evidence from other V. vulnificus enzymes indicates that periplasmic proteins may be active only in their oxidized form but not in their reduced form . This suggests that disulfide bond formation, which is facilitated in the oxidizing environment of the periplasm, may be critical for ddl function and stability.

• Metal ion availability: The periplasmic space has different metal ion concentrations compared to the cytoplasm, potentially affecting metal-dependent enzymes like ddl, which typically requires divalent cations (usually Mg²⁺) for catalysis.

• pH considerations: The periplasm can experience pH fluctuations different from the cytoplasm, particularly under stress conditions. This may affect enzyme kinetics and stability, potentially modulating resistance properties.

• Spatial compartmentalization: Periplasmic localization may provide strategic advantages for resistance determinants by providing a first line of defense against antimicrobials that must traverse the periplasm to reach their targets or by concentrating the enzyme where it's needed for cell wall modification.

• Protein-protein interactions: The periplasmic environment may facilitate interactions with other components of the cell wall synthesis machinery, potentially enhancing the efficiency of resistance mechanisms.

For integron-encoded ddl variants specifically, their periplasmic localization (if confirmed) could be particularly significant for resistance functions, as modifications to peptidoglycan precursors in this compartment would directly impact cell wall structure and antibiotic interactions before these compounds reach their targets.

How can understanding Vibrio vulnificus ddl variants contribute to the development of new antibiotics?

Understanding the structure, function, and resistance mechanisms of Vibrio vulnificus ddl variants offers several avenues for antibiotic development:

• Structure-based drug design: Detailed structural characterization of these enzymes, particularly their unique features compared to other ddl variants, could enable the design of selective inhibitors that overcome resistance mechanisms. Special attention to the omega-loop region may reveal targetable differences from human enzymes.

• Resistance-proof targeting: The finding that single mutations can alter resistance profiles highlights the importance of designing inhibitors that target conserved, functionally essential residues where mutations would severely compromise enzyme function, raising the genetic barrier to resistance development.

• Combination strategies: Knowledge of how these ddl variants confer D-cycloserine resistance could inform the development of adjuvants that restore sensitivity to existing antibiotics, potentially extending the useful life of the current antibiotic arsenal.

• Natural product inspiration: The hypothesis linking dietary flavonoids to ddl inhibition suggests exploring natural flavonoids and their derivatives as starting points for developing novel ddl inhibitors with potentially favorable pharmacokinetic properties.

• Cross-species targeting: The distinct phylogenetic position of these ddl variants suggests they may have unique features compared to ddl enzymes in other pathogens, potentially enabling the development of narrow-spectrum antibiotics specific to Vibrio species.

• Integron-targeting approaches: Understanding the genetic context of these resistance determinants opens possibilities for developing therapeutics that destabilize integrons or prevent gene cassette expression, indirectly combating resistance mechanisms that rely on these mobile genetic elements.

What experimental models are most appropriate for studying the role of ddl in Vibrio vulnificus virulence?

While the search results indicate that some Vibrio vulnificus factors (like the nuclease Vvn) are not required for virulence in mice , designing appropriate experimental models for studying ddl's potential role in pathogenesis requires careful consideration:

• In vitro cellular models:

  • Human intestinal epithelial cell lines to study colonization and invasion

  • Macrophage infection models to assess intracellular survival

  • Neutrophil killing assays to evaluate resistance to innate immunity

  • Tissue explant models to simulate complex barrier functions

• Animal infection models:

  • Mouse peritoneal infection model for systemic virulence assessment

  • Iron-overloaded or immunocompromised mouse models to mimic susceptible human hosts

  • Eel models for environmental host interactions

  • Specialized models like suction blister skin models for wound infection studies

• Genetic manipulation strategies:

  • Clean deletion mutants of specific ddl variants to assess their contribution to virulence

  • Complementation studies to confirm phenotype specificity

  • Site-directed mutants with altered resistance properties but maintained catalytic function

  • Conditional expression systems to study temporal requirements during infection

• Measurement parameters:

  • Bacterial burden in tissues

  • Inflammatory marker production

  • Tissue damage assessment

  • Survival curves under antibiotic treatment

  • Peptidoglycan structure analysis during infection

How might the flavonoid-ddl interaction hypothesis inform alternative approaches to controlling antibiotic resistance?

The hypothesis that dietary flavonoids with Ddl inhibitory activity could act as a driver for selecting ddls within integrons presents intriguing possibilities for alternative resistance control strategies:

• Dietary intervention approaches:

  • Identifying specific flavonoids that inhibit resistant ddl variants

  • Developing dietary supplements that potentiate antibiotic activity

  • Exploring traditional diets rich in flavonoids for natural resistance-modulating compounds

• Combination therapy potential:

  • Using flavonoid derivatives as adjuvants alongside conventional antibiotics

  • Developing dual-targeting drugs that incorporate flavonoid-like moieties with other antimicrobial components

  • Exploring synergistic effects between flavonoids and antibiotics targeting different steps in cell wall synthesis

• Resistance evolution management:

  • Using flavonoids to suppress emergence of resistant populations

  • Alternating or combining flavonoid-based compounds with conventional antibiotics to reduce selection pressure

  • Targeting integron stability with flavonoid derivatives to reduce horizontal gene transfer

• Environmental intervention:

  • Modifying aquaculture practices to include flavonoid-rich plant materials

  • Developing environmental treatments for water systems where Vibrio species are problematic

  • Creating flavonoid-enriched biofilms or surfaces in high-risk environments

• Screening and development pipeline:

  • Using flavonoid scaffolds as starting points for developing novel ddl inhibitors

  • Creating libraries of flavonoid derivatives optimized for specificity against resistant ddl variants

  • Developing high-throughput screening methods using resistant ddl variants to identify effective inhibitors

This hypothesis connects natural products chemistry with resistance biology in a way that could lead to more sustainable approaches to resistance management, potentially reducing reliance on conventional antibiotics while targeting resistance mechanisms directly.

What bioinformatic approaches are most effective for identifying and analyzing novel ddl variants in bacterial genomes?

Comprehensive identification and characterization of novel ddl variants requires sophisticated bioinformatic strategies:

• Sequence-based identification approaches:

  • Profile hidden Markov models (HMMs) built from known ddl sequences, including the divergent integron-encoded variants

  • Position-specific scoring matrices that account for conserved catalytic residues

  • BLAST searches using representatives from different ddl clades as queries

  • Synteny-based searches that consider genomic context, particularly integron-associated genes

• Functional annotation refinement:

  • Active site motif identification to distinguish true ddl enzymes from other ATP-grasp fold proteins

  • Substrate specificity prediction based on binding site residues

  • Resistance phenotype prediction using machine learning models trained on known resistance-conferring variants

  • Integration of expression data to identify functionally relevant variants

• Evolutionary analysis tools:

  • Phylogenetic reconstruction methods that can accurately place highly divergent sequences

  • Selection pressure analysis to identify residues under positive selection

  • Horizontal gene transfer detection algorithms to identify recently acquired ddl genes

  • Ancestral sequence reconstruction to infer evolutionary trajectories

• Structural bioinformatics:

  • Homology modeling to predict structures of novel variants

  • Molecular dynamics simulations to assess stability and flexibility differences

  • Protein-ligand docking to predict interactions with antibiotics and inhibitors

  • Structural motif mining to identify features shared with vancomycin resistance proteins

• Integron-specific approaches:

  • Specialized algorithms for detecting integron structures and gene cassettes

  • Promoter strength prediction to assess potential expression levels in different positions

  • Attenuator structure identification to predict post-transcriptional regulation

  • Codon usage analysis to identify genes optimized for expression in specific hosts

These approaches should be implemented as part of a comprehensive pipeline that integrates genomic, structural, and functional data to provide a complete characterization of novel ddl variants and their potential clinical significance.

How can the interaction between ddl mutations and antibiotic resistance be experimentally validated in Vibrio vulnificus?

Rigorous experimental validation of the relationship between ddl mutations and antibiotic resistance requires a multi-faceted approach:

• Genetic manipulation strategies:

  • CRISPR-Cas9 genome editing to introduce specific mutations into the native ddl gene

  • Allelic exchange methods to replace wild-type ddl with mutant variants

  • Heterologous expression systems to test mutant ddl variants in isolation

  • Complementation studies in ddl-knockout strains to confirm phenotype causality

• Resistance phenotype characterization:

  • Standardized MIC determination using broth microdilution according to CLSI guidelines

  • Time-kill kinetics to assess the dynamics of antibiotic action

  • Population analysis profiling to detect heteroresistance

  • In vivo efficacy testing in animal infection models

• Mechanistic investigations:

  • Enzyme kinetics comparing wild-type and mutant proteins under physiologically relevant conditions

  • Thermal shift assays to assess changes in protein stability

  • Isothermal titration calorimetry to measure binding affinities for substrates and inhibitors

  • Structural studies using X-ray crystallography or cryo-EM to visualize molecular changes

• Comprehensive validation approaches:

  • Whole-genome sequencing to identify potential compensatory mutations

  • Transcriptome analysis to assess global regulatory effects

  • Peptidoglycan structure analysis using mass spectrometry

  • Fitness cost determination through competition assays

• Translational validation:

  • Testing against clinical isolates harboring similar mutations

  • Surveillance studies to determine mutation prevalence in clinical settings

  • Assessment of cross-resistance to other antibiotics

  • Evolution experiments to determine resistance development trajectories

What cutting-edge technologies could advance our understanding of D-alanine-D-alanine ligase function in antimicrobial resistance?

Several emerging technologies offer promising approaches to deepen our understanding of ddl function and resistance mechanisms:

• Advanced structural biology techniques:

  • Cryo-electron microscopy for visualizing enzyme-substrate complexes without crystallization

  • Micro-electron diffraction for structural determination from nanocrystals

  • Time-resolved X-ray crystallography to capture catalytic intermediates

  • Nuclear magnetic resonance studies of protein dynamics in solution

• High-resolution cellular imaging:

  • Super-resolution microscopy to visualize ddl localization during cell wall synthesis

  • Correlative light and electron microscopy to connect enzyme activity with ultrastructural changes

  • Single-molecule tracking to assess enzyme mobility and interactions in living cells

  • Expansion microscopy to visualize peptidoglycan synthesis machinery

• Advanced genetic manipulation tools:

  • Base editing technologies for precise single nucleotide modifications

  • CRISPRi/CRISPRa for reversible modulation of ddl expression

  • CRISPR-scanning for high-throughput functional mapping of ddl domains

  • Synthetic genomics approaches to create minimized systems for studying essential functions

• Systems biology approaches:

  • Multi-omics integration combining genomics, transcriptomics, proteomics, and metabolomics

  • Flux analysis of peptidoglycan precursors under antibiotic pressure

  • Network analysis of cell wall synthesis pathway interactions

  • Machine learning for predicting resistance phenotypes from sequence features

• Innovative screening technologies:

  • Microfluidic single-cell analysis of antibiotic response heterogeneity

  • Droplet-based high-throughput screening for inhibitor discovery

  • Deep mutational scanning to comprehensively map mutation effects

  • Cell-free expression systems for rapid functional assessment

• Advanced computational methods:

  • Quantum mechanics/molecular mechanics simulations of catalytic mechanisms

  • Free energy perturbation calculations for precise binding affinity predictions

  • Artificial intelligence-driven drug design targeting resistant ddl variants

  • Metadynamics simulations to identify cryptic binding sites

These cutting-edge approaches, particularly when integrated in complementary combinations, have the potential to revolutionize our understanding of ddl function in antibiotic resistance and accelerate the development of novel therapeutic strategies to combat resistance mechanisms in Vibrio vulnificus and other pathogens.