Recombinant Aeromonas salmonicida Nucleoside diphosphate kinase (ndk)

What is the fundamental function of ndk in Aeromonas salmonicida?

Nucleoside diphosphate kinase in A. salmonicida serves a major role in the synthesis of nucleoside triphosphates other than ATP. The enzyme transfers the gamma phosphate from ATP to the beta phosphate of nucleoside diphosphates (NDPs) via a ping-pong mechanism, utilizing a phosphorylated active-site intermediate . This catalytic activity is essential for maintaining nucleotide pools necessary for DNA replication, RNA synthesis, and various signaling pathways during bacterial growth and infection processes.

How is the ndk protein structurally characterized in Aeromonas species?

The ndk protein in Aeromonas species is typically a small protein (approximately 140-150 amino acids) that forms oligomeric structures, most commonly hexamers. Based on comparative analysis with the E. coli ndk, the protein likely contains a conserved catalytic core featuring a Rossmann fold characteristic of nucleotide-binding proteins and a critical histidine residue that becomes phosphorylated during the catalytic cycle . The high degree of genetic conservation observed among A. salmonicida isolates suggests that the ndk protein sequence is likely highly preserved across different strains .

What expression systems are most effective for producing recombinant A. salmonicida ndk?

For laboratory-scale production of recombinant A. salmonicida ndk, E. coli-based expression systems have proven most effective. Similar to the approach used for E. coli ndk, researchers typically employ vectors containing hexahistidine tags to facilitate purification . The optimal expression conditions include:

What purification strategies yield the highest purity A. salmonicida ndk protein?

The most effective purification strategy for recombinant A. salmonicida ndk involves a multi-step approach:

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin for initial capture of His-tagged protein

• Ion exchange chromatography to remove nucleic acid contaminants and similarly charged proteins

• Size exclusion chromatography as a polishing step to achieve >95% purity and remove any aggregates

This approach has been successfully employed for E. coli ndk and is likely transferable to A. salmonicida ndk due to their similar biochemical properties . The purified protein should be confirmed via SDS-PAGE and mass spectrometry to verify integrity and purity.

How can researchers assess the enzymatic activity of purified recombinant ndk?

Several robust methodologies exist for assessing ndk activity:

What are the critical factors for maintaining stability of recombinant A. salmonicida ndk during storage?

Based on general protein biochemistry principles and data from similar enzymes, optimal storage conditions include:

• Buffer composition: 50 mM Tris-HCl or HEPES (pH 7.5), 100-150 mM NaCl, 5 mM MgCl₂ (essential cofactor), 1 mM DTT (to prevent oxidation), 10% glycerol

• Temperature: -80°C for long-term storage; -20°C with 50% glycerol for medium-term; 4°C for short-term (1-2 weeks)

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Addition of protease inhibitors for preparations that will undergo further analysis

How does A. salmonicida ndk potentially contribute to bacterial virulence?

While the specific role of ndk in A. salmonicida virulence has not been extensively characterized, research on related bacterial pathogens suggests several potential mechanisms:

• Immune modulation: ndk may interfere with host cell signaling pathways, particularly those involving GTP-binding proteins

• Survival under stress: ndk contributes to nucleotide homeostasis during infection, potentially allowing adaptation to host environments

• Secretion: Some bacterial ndks can be secreted and interact directly with host cell components

• Contribution to virulence factor expression: Optimal nucleotide pools maintained by ndk may regulate expression of virulence genes

Given that A. salmonicida possesses multiple virulence factors (including ascV, fla, ahyB, gcaT, lip, alt, and act as identified in search result ), research investigating potential interactions between ndk and these virulence systems could be particularly valuable.

What role might ndk play in antimicrobial resistance in A. salmonicida?

The emergence of multidrug-resistant A. salmonicida strains raises questions about ndk's potential role in resistance mechanisms:

• Metabolic adaptation: ndk may help maintain nucleotide pools necessary for expression of resistance genes

• Biofilm formation: Potential involvement in signaling pathways that regulate biofilm development, which can enhance antibiotic tolerance

• Stress response: Contribution to nucleotide homeostasis during antibiotic stress, potentially enhancing survival

Research examining differential expression of ndk in resistant versus susceptible isolates could provide insights into these potential relationships.

How might ndk function interact with bacteriophage infection processes?

Given the increasing interest in phage therapy for controlling A. salmonicida infections , understanding ndk's role during phage infection is relevant:

• Nucleotide provision: ndk activity may be essential for providing nucleotides needed for phage replication

• Host-phage interactions: ndk may be targeted or modulated by phage proteins during infection

• Restriction-modification systems: ndk may indirectly support DNA modification systems that protect against phage infection

The characterization of phages like JELG-KS1 and ASP-1 that infect A. salmonicida provides experimental systems to investigate these potential interactions.

How does A. salmonicida ndk compare structurally and functionally with ndk from other bacterial species?

Comparative analysis reveals important insights into conservation and specialization:

*Predicted based on related species and A. salmonicida habitat requirements

What evolutionary pressures have shaped ndk in A. salmonicida compared to other Aeromonas species?

Genomic analysis of A. salmonicida reveals evidence of evolutionary adaptation to its salmonid host environment . For ndk, these adaptations likely include:

• Temperature adaptation: Modifications for optimal activity at lower temperatures typical of aquatic environments

• Host-specific adaptations: Potential refinements related to nucleotide availability in fish hosts

• Selective pressure from bacteriophages: Co-evolutionary relationships with phages that target A. salmonicida

• Conservation of core function: The essential metabolic role of ndk likely constrains radical evolutionary changes

The high degree of genetic conservation observed among A. salmonicida isolates from different outbreaks suggests that ndk, as a core metabolic gene, likely maintains high sequence conservation.

How can recombinant A. salmonicida ndk contribute to vaccine development against furunculosis?

With rising antimicrobial resistance concerns in aquaculture , ndk presents several opportunities for vaccine development:

• Subunit vaccine component: Recombinant ndk could be included in multicomponent vaccines targeting multiple A. salmonicida antigens

• Carrier protein: ndk could be engineered as a carrier for other epitopes to enhance immunogenicity

• DNA vaccine: Genetic immunization with ndk-encoding constructs

• Target identification: Structural studies of ndk could reveal epitopes for rational vaccine design

The high conservation of ndk across A. salmonicida isolates makes it potentially valuable for broad-spectrum protection, though its intracellular localization might limit effectiveness unless combined with surface antigens.

What is the potential for ndk as a drug target for controlling A. salmonicida infections?

As an essential enzyme, ndk represents a potential therapeutic target:

How might recombinant ndk be utilized in developing phage-based therapies against A. salmonicida?

The emerging field of phage therapy for A. salmonicida infections could benefit from ndk-related approaches:

• Combination therapies: ndk inhibitors combined with phage treatment to reduce resistance development

• Phage engineering: Modification of phages to target ndk-dependent processes

• Biofilm disruption: Targeting ndk-mediated processes involved in biofilm formation, enhancing phage access

• Resistance monitoring: Using ndk activity as a biomarker for metabolic shifts during phage resistance development

Studies examining A. salmonicida adaptation during phage infection, particularly with characterized phages like JELG-KS1 and ASP-1 , could reveal valuable insights for these applications.

What advanced techniques are most informative for structural characterization of A. salmonicida ndk?

Multiple complementary approaches provide comprehensive structural insights:

• X-ray crystallography: For high-resolution atomic structure determination, particularly of the active site and substrate binding pocket

• Cryo-electron microscopy: For visualization of oligomeric assembly and conformational states

• Hydrogen-deuterium exchange mass spectrometry: For mapping protein dynamics and ligand-induced conformational changes

• Small-angle X-ray scattering (SAXS): For solution-state analysis of oligomeric structure

• Nuclear magnetic resonance (NMR): For studying protein-ligand interactions and dynamics in solution

What experimental approaches can resolve contradictory findings about ndk function in different A. salmonicida strains?

When faced with contradictory results, researchers should implement:

The solid-state nanopore sequencing (ONT) approach described for genomic analysis of A. salmonicida isolates provides a powerful tool for identifying strain-specific variations that might explain functional differences.

What emerging technologies could advance understanding of A. salmonicida ndk's role in pathogenesis?

Several cutting-edge approaches show promise:

• CRISPR-Cas9 gene editing: For precise modification of ndk in A. salmonicida to study function

• Single-cell transcriptomics: To examine ndk expression heterogeneity during infection

• Cryo-electron tomography: For visualizing ndk localization and interactions in intact bacterial cells

• Interactome mapping: Identifying protein-protein interactions involving ndk using proximity labeling approaches

• Metabolomics: Analyzing nucleotide pools and flux in wild-type versus ndk-modified strains

How might climate change impact ndk function in A. salmonicida and what research approaches can address this question?

As aquatic environments change, A. salmonicida may face new selective pressures affecting ndk function:

• Temperature adaptation: How will warming waters affect the activity of cold-adapted ndk?

• Host range expansion: Will ndk play a role in adaptation to new fish hosts as species distributions shift?

• Interaction with emerging pathogens: Potential competitive or synergistic effects with other microorganisms

Research approaches should include experimental evolution studies under simulated climate change conditions, comparative genomics across geographical isolates, and thermal stability analysis of recombinant ndk variants.

What integrative approaches could best reveal ndk's role in the complex host-pathogen-phage interactions in aquaculture settings?

Understanding the complex ecological interactions requires multidisciplinary approaches:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data

• Mathematical modeling: Developing predictive models of ndk function in different ecological scenarios

• Field studies: Examining ndk sequence variation and expression in natural outbreaks

• Host-pathogen co-culture systems: Analyzing ndk regulation during interaction with fish cells

• Microbiome studies: Investigating how ndk contributes to A. salmonicida's interactions with the fish microbiome