Recombinant Aliivibrio salmonicida Nucleoside diphosphate kinase (ndk)

What is Aliivibrio salmonicida and what is its significance in research?

Aliivibrio salmonicida is a gram-negative, rod-shaped bacterium with flagella that belongs to the family Vibrionaceae. It is the causative agent of cold-water vibriosis (CV), a hemorrhagic septicemia that affects farmed Atlantic salmon (Salmo salar), sea-farmed rainbow trout (Oncorhynchus mykiss), and captive Atlantic cod (Gadus morhua) . The bacterium was previously known as Vibrio salmonicida before taxonomic reclassification .

A. salmonicida has significant research importance due to its pathogenic nature and economic impact on aquaculture, particularly in countries like Norway. The bacterium rapidly enters the fish bloodstream, with proliferation occurring after a period of latency . Understanding the molecular mechanisms of A. salmonicida pathogenesis is crucial for developing effective control strategies for cold-water vibriosis in aquaculture settings.

What are nucleoside diphosphate kinases and what functions do they serve?

Nucleoside diphosphate kinases (NDKs) are evolutionarily conserved enzymes found across all organisms that play crucial roles in numerous cellular processes. Their primary function is maintaining intracellular di- and tri-phosphate nucleoside homeostasis through the following reaction:

Nucleoside triphosphate (NTP) + Nucleoside diphosphate (NDP) → Nucleoside diphosphate (NDP) + Nucleoside triphosphate (NTP)

The catalysis involves phosphorylation of a conserved histidine residue which subsequently phosphorylates the NDP through a ping-pong mechanism .

Beyond their canonical role in nucleotide metabolism, NDKs are multifunctional enzymes involved in diverse processes including gene regulation, apoptosis, signal transduction, and various other regulatory functions . This multifunctionality makes NDKs particularly interesting targets for both basic research and potential therapeutic applications.

How is recombinant NDK typically expressed and purified for research purposes?

The expression and purification of recombinant A. salmonicida NDK typically involves molecular cloning techniques similar to those used for other bacterial NDKs. The general methodology includes:

• PCR amplification of the ndk gene from A. salmonicida genomic DNA with appropriate primers containing restriction sites

• Cloning into an expression vector (commonly pET-based systems for bacterial expression)

• Transformation into a suitable E. coli expression strain (BL21(DE3) or derivatives)

• Induction of protein expression using IPTG or auto-induction methods

• Cell lysis using sonication or commercial lysis buffers

• Purification using affinity chromatography (typically His-tag purification)

• Further purification by size exclusion or ion exchange chromatography if needed

• Verification of purity by SDS-PAGE and protein identification by Western blot and/or mass spectrometry

Purified recombinant NDK can be separated on a 15% SDS-PAGE gel for analysis . Researchers must optimize expression conditions including temperature, induction time, and IPTG concentration to maximize yield and solubility of the recombinant protein.

What experimental approaches can be used to assess the enzymatic activity of recombinant NDK?

Several robust methodologies can be employed to evaluate the enzymatic activity of recombinant A. salmonicida NDK:

• Coupled enzyme assays: NDK activity can be measured by coupling ATP generation to a secondary enzyme reaction such as the luciferase-luciferin system, which produces luminescence proportional to ATP concentration.

• Direct phosphate transfer assays: Using radiolabeled nucleotides (such as [γ-32P]ATP) to measure the transfer of radioactive phosphate to nucleoside diphosphates.

• HPLC analysis: Quantifying the conversion of nucleoside diphosphates to nucleoside triphosphates through high-performance liquid chromatography.

• Isothermal titration calorimetry (ITC): As demonstrated with other NDKs, ITC can be used to measure nucleotide binding affinity. For example, recombinant NDK has been shown to bind ADP and GDP with dissociation constants (Kd) of 153 and 157 μmol/liter, respectively, while showing no significant binding to CDP and UDP .

• Malachite green phosphate assay: Measuring the release of inorganic phosphate during NDK-catalyzed reactions.

For meaningful interpretation of results, appropriate positive controls (such as commercially available NDKs) and negative controls (heat-inactivated enzyme) should be included in all enzymatic assays.

How can researchers investigate the nucleotide binding properties of A. salmonicida NDK?

Investigation of nucleotide binding properties is critical for understanding NDK function. Researchers can employ the following techniques:

• Isothermal Titration Calorimetry (ITC): ITC provides direct measurement of binding thermodynamics. As shown with similar NDKs, this method can determine binding affinities for various nucleotides (ADP, GDP, CDP, UDP). The binding occurs with dissociation constants typically in the micromolar range (e.g., Kd of 153 μmol/liter for ADP and 157 μmol/liter for GDP) .

• Surface Plasmon Resonance (SPR): This technique allows real-time analysis of protein-nucleotide interactions without labeling requirements.

• Fluorescence-based assays: Using intrinsic tryptophan fluorescence or extrinsic fluorophores to detect conformational changes upon nucleotide binding.

• X-ray crystallography: Determining the three-dimensional structure of NDK in complex with different nucleotides to identify binding residues and conformational changes.

• NMR spectroscopy: For investigating dynamic aspects of nucleotide binding and potential allosteric effects.

When investigating nucleotide specificity, researchers should examine a panel of nucleotides including ATP, GTP, CTP, UTP and their corresponding diphosphate forms to establish a comprehensive binding profile.

What approaches are recommended for studying potential multifunctionality of A. salmonicida NDK?

NDKs are known for their multifunctionality beyond canonical nucleotide metabolism . To investigate these potential alternative functions in A. salmonicida NDK, researchers should consider:

• Protein-protein interaction studies:

  • Pull-down assays with cell lysates to identify binding partners

  • Yeast two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Bacterial two-hybrid systems specifically adapted for bacterial proteins

• DNA/RNA interaction analysis:

  • Electrophoretic mobility shift assays (EMSA)

  • Chromatin immunoprecipitation (for potential gene regulatory roles)

  • RNA immunoprecipitation (RIP) for RNA-binding capabilities

• Phosphotransferase activity beyond nucleotides:

  • Assays with non-nucleotide substrates like proteins and lipids

  • In vitro phosphorylation assays with potential target proteins

• Comparative functional genomics:

  • Analysis of phenotypic changes in NDK knockout mutants

  • Complementation studies with NDKs from different species

  • Transcriptomic analysis of NDK-deficient versus wild-type bacteria

• Subcellular localization studies:

  • Immunofluorescence microscopy with anti-NDK antibodies

  • Fractionation studies to determine distribution across bacterial compartments

These approaches should be conducted in parallel to build a comprehensive understanding of A. salmonicida NDK's functional repertoire.

How can researchers generate and validate NDK-deficient A. salmonicida mutants?

Creating NDK-deficient mutants is essential for studying the role of NDK in A. salmonicida pathogenesis. Based on methodologies used for similar genetic manipulations in A. salmonicida, researchers can follow these approaches:

• Allelic exchange mutagenesis using suicide plasmids:

  • Amplify the upstream and downstream regions flanking the ndk gene

  • Clone these regions into a suicide vector (e.g., pDM4 or pFS100)

  • Transfer the recombinant plasmid into A. salmonicida via triparental mating using a mobilizing strain such as HB101/pRK2073

  • Select transconjugants on appropriate antibiotic media

  • Counter-select for second crossover events using sucrose sensitivity (for sacB-containing vectors)

  • Confirm the deletion by PCR and sequencing

• Insertional inactivation:

  • Amplify an internal fragment of the ndk gene

  • Clone this fragment into a suicide vector like pFS100

  • Transfer into A. salmonicida by triparental mating

  • Select for single crossover insertions that disrupt the gene

• CRISPR-Cas9 based approaches for more precise gene editing

Validation of mutants should include:

• PCR verification of gene deletion/disruption

• RT-qPCR to confirm absence of ndk transcript

• Western blot to confirm absence of NDK protein

• Enzymatic assays to confirm loss of NDK activity

• Complementation studies to restore wild-type phenotype

• Whole genome sequencing to ensure no off-target mutations

These experimental approaches ensure the creation of reliable mutants for downstream functional studies.

What experimental models are appropriate for investigating the role of NDK in A. salmonicida virulence?

To investigate the role of NDK in A. salmonicida virulence, researchers should consider multiple experimental models:

• In vitro models:

  • Bacterial survival assays in fish serum

  • Adhesion and invasion assays using fish cell lines

  • Biofilm formation assays

  • Resistance to oxidative stress and antimicrobial peptides

  • Bacterial growth kinetics under various conditions

• Ex vivo models:

  • Primary fish tissue cultures

  • Organ explants from susceptible fish species

• In vivo models:

  • Immersion challenge in Atlantic salmon (Salmo salar) or other susceptible species

  • Intraperitoneal injection for direct introduction of bacteria

  • Quantification of bacteria in blood following challenge

  • Monitoring immune responses in challenged fish

When designing these experiments, it is crucial to include:

• Wild-type A. salmonicida as positive control

• NDK-deficient mutants

• Complemented mutants (NDK-deficient strains with restored NDK expression)

• Appropriate environmental controls (temperature, pH, salinity)

For in vivo studies, track parameters such as mortality rates, bacterial load in tissues, histopathological changes, and immune response markers. The research by Bjelland et al. demonstrated that O-antigen deficient mutants of A. salmonicida induced a more transient immune response than wild-type strains, suggesting a similar approach could be valuable for NDK studies .

What controls and considerations are essential when analyzing the enzymatic properties of recombinant A. salmonicida NDK?

When analyzing enzymatic properties of recombinant A. salmonicida NDK, researchers must implement rigorous controls and considerations:

• Essential controls:

  • Positive control: commercially available NDK with known activity

  • Negative controls: heat-inactivated enzyme and reaction mixture without enzyme

  • Buffer controls to account for non-enzymatic nucleotide conversion

  • Substrate controls to ensure stability throughout the experiment

• Enzymatic activity considerations:

  • Optimal temperature determination (considering A. salmonicida is a cold-water pathogen)

  • pH optimization within physiologically relevant ranges

  • Metal ion dependency (typically Mg2+ or Mn2+)

  • Substrate specificity across different nucleotide pairs

  • Kinetic parameters (Km, Vmax, kcat) for various substrates

• Protein quality considerations:

  • Purity assessment (>95% recommended for kinetic studies)

  • Verification of proper folding using circular dichroism

  • Stability testing at different temperatures and storage conditions

  • Batch-to-batch consistency validation

• Assay validation:

  • Linear range determination

  • Reproducibility across technical and biological replicates

  • Limit of detection and quantification

• Data analysis considerations:

  • Appropriate kinetic models for data fitting

  • Statistical analysis of replicate experiments

  • Comparison with published data on related NDKs

Following these guidelines ensures reliable and reproducible characterization of recombinant A. salmonicida NDK enzymatic properties.

How can researchers investigate potential structural features of A. salmonicida NDK?

Understanding the structural features of A. salmonicida NDK is essential for elucidating its function and developing potential inhibitors. Researchers can employ the following approaches:

• Protein structure prediction and analysis:

  • Homology modeling based on related NDK structures

  • Molecular dynamics simulations to study protein flexibility

  • Binding site prediction for nucleotides and other ligands

  • Identification of conserved catalytic residues, particularly the phosphohistidine site

• Experimental structure determination:

  • X-ray crystallography of purified recombinant protein

  • NMR spectroscopy for solution structure and dynamics

  • Cryo-electron microscopy for quaternary structure analysis

• Structure-function relationship studies:

  • Site-directed mutagenesis of predicted catalytic residues

  • Analysis of oligomeric state (NDKs typically form hexamers)

  • Thermal stability assays (DSF/DSC) to evaluate structural integrity

• Structural comparison with NDKs from other species:

  • Sequence alignment and structural superposition

  • Conservation analysis of binding sites

  • Identification of unique structural features in A. salmonicida NDK

The structural analysis should focus on the active site histidine residue involved in the phosphorylation mechanism and the nucleotide binding pocket, which affects substrate specificity as demonstrated in related NDK studies .

What methodologies can assess the potential of A. salmonicida NDK as a drug target?

To evaluate A. salmonicida NDK as a potential drug target for treating cold-water vibriosis, researchers should implement a systematic approach:

• Target validation studies:

  • Essentiality assessment using conditional mutants

  • Phenotypic characterization of NDK-deficient strains

  • In vivo virulence studies comparing wild-type and NDK-deficient mutants

  • Complementation studies to confirm phenotype restoration

• Structural and biochemical characterization:

  • High-resolution structural determination of A. salmonicida NDK

  • Identification of unique features compared to host (fish) NDKs

  • Enzymatic assays to establish baseline kinetic parameters

  • Substrate specificity profiling

• Inhibitor discovery and development:

  • High-throughput screening of compound libraries

  • Fragment-based drug discovery approaches

  • Structure-based virtual screening

  • Development of biochemical and cell-based assays for inhibitor testing

• Lead compound characterization:

  • Structure-activity relationship studies

  • Binding affinity determination using ITC or SPR

  • Selectivity profiling against host NDKs

  • Cellular uptake and cytotoxicity assessment

• Preclinical evaluation:

  • In vitro efficacy against A. salmonicida cultures

  • Ex vivo testing in relevant fish tissue models

  • Pharmacokinetic studies in fish species

  • Preliminary toxicity assessment

This approach aligns with the growing recognition of NDKs as potential targets for therapeutic intervention in bacterial pathogens, as highlighted in studies of trypanosomatid NDKs .

How do researchers analyze data inconsistencies when working with recombinant A. salmonicida NDK?

When confronted with data inconsistencies in recombinant A. salmonicida NDK research, scientists should follow these systematic troubleshooting approaches:

• Protein quality assessment:

  • Verify protein purity using multiple methods (SDS-PAGE, size exclusion chromatography)

  • Check for proper folding using circular dichroism or intrinsic fluorescence

  • Assess protein stability under experimental conditions

  • Verify the absence of contaminating enzymatic activities

• Experimental design evaluation:

  • Review experimental controls for adequacy

  • Ensure appropriate statistical power through sufficient replication

  • Validate assay linearity, sensitivity, and reproducibility

  • Consider potential interfering factors in assay components

• Data analysis approaches:

  • Apply appropriate statistical tests for the data distribution

  • Identify and handle outliers based on objective criteria

  • Use multiple analytical methods and compare results

  • Consider blinding data analysis to prevent bias

• Reconciling conflicting data:

  • Compare with published literature on related NDKs

  • Consider differences in experimental conditions that may explain discrepancies

  • Evaluate the effect of post-translational modifications or alternate isoforms

  • Design experiments specifically to address contradictions

• Technical considerations:

  • Calibrate all instruments regularly

  • Use multiple batches of recombinant protein to account for batch variation

  • Consider the impact of freeze-thaw cycles on enzyme activity

  • Standardize protocols across experiments and researchers

This methodical approach helps researchers identify sources of inconsistency and develop reliable experimental protocols for working with recombinant A. salmonicida NDK.

How should researchers interpret nucleotide binding data for A. salmonicida NDK?

When interpreting nucleotide binding data for A. salmonicida NDK, researchers should consider these key aspects:

• Binding affinity analysis:

  • Compare dissociation constants (Kd) across different nucleotides

  • For related NDKs, binding affinities for ADP and GDP are typically in the range of 150-160 μmol/liter

  • Analyze thermodynamic parameters (ΔH, ΔS, ΔG) to understand the nature of binding

• Specificity considerations:

  • Evaluate binding across the complete panel of physiologically relevant nucleotides

  • Assess preference patterns (purine vs. pyrimidine nucleotides)

  • Compare with binding profiles of NDKs from other species

• Structure-function relationships:

  • Correlate binding data with structural features

  • Consider the impact of conserved residues in the binding pocket

  • Evaluate the role of divalent cations (typically Mg2+) in nucleotide binding

• Physiological context:

  • Consider intracellular nucleotide concentrations in A. salmonicida

  • Evaluate potential regulatory mechanisms affecting binding

  • Assess the impact of environmental conditions (temperature, pH) on binding properties

• Data presentation:

  • Present binding data in standardized formats (tables with Kd values)

  • Include representative binding curves

  • Provide statistical analysis of replicate experiments

The following table format can be used to present nucleotide binding data:

*Based on data from related NDKs

What future research directions should be prioritized for A. salmonicida NDK studies?

For advancing A. salmonicida NDK research, the following priorities are recommended:

• Comprehensive structural characterization:

  • Determine high-resolution crystal structures in various nucleotide-bound states

  • Analyze oligomeric assembly and potential regulatory mechanisms

  • Investigate potential post-translational modifications

• Systems biology approaches:

  • Transcriptomic and proteomic analysis of NDK-deficient mutants

  • Global metabolomic profiling to assess impact on nucleotide homeostasis

  • Network analysis to identify functional relationships with other cellular components

• Host-pathogen interaction studies:

  • Investigate potential roles in immune evasion

  • Assess impact on host cell signaling pathways

  • Examine potential secretion of NDK during infection

• Environmental adaptation mechanisms:

  • Study NDK function under various temperature and pH conditions relevant to aquaculture

  • Investigate potential roles in stress response and adaptation

  • Analyze expression patterns under different growth conditions

• Translational research:

  • Development of NDK-based vaccines for aquaculture

  • High-throughput screening for selective inhibitors

  • Engineering NDK variants for potential biotechnological applications

• Comparative studies:

  • Compare enzymatic properties with NDKs from related fish pathogens

  • Analyze functional conservation and divergence across Vibrionaceae

  • Study co-evolution with host species

These research directions would significantly advance understanding of A. salmonicida NDK biology and potentially lead to new strategies for controlling cold-water vibriosis in aquaculture.

How can copy number variation of NDK be accurately determined in A. salmonicida?

Accurate determination of NDK copy number in A. salmonicida is crucial for genetic studies and mutant validation. Researchers should consider these methodological approaches:

• Quantitative PCR (qPCR) based methods:

  • Design primers specific to ndk and reference genes

  • Use standard curves for absolute quantification

  • Apply the 2-ΔΔCt method for relative quantification

  • Include multiple reference genes for normalization

• Digital PCR for absolute copy number determination:

  • Provides higher precision for copy number analysis

  • Less susceptible to PCR inhibitors and efficiency variations

  • Allows absolute quantification without standard curves

• Whole genome sequencing approaches:

  • Calculate depth of coverage across the genome

  • Compare ndk coverage relative to single-copy genes

  • Analyze read depth ratios for copy number estimation

• Southern blotting:

  • Use ndk-specific probes to visualize gene copies

  • Include restriction digest analysis for confirmation

  • Compare band intensities for semi-quantitative analysis

• Data analysis and presentation:

  • Calculate mean copy number with 95% confidence intervals

  • Round to nearest integer for final copy number determination

  • Present data in tabular format similar to the example below

Example format for presenting copy number data:

This table format, adapted from similar gene copy number analyses , provides a clear presentation of copy number data with appropriate statistical measures.