Recombinant Aliivibrio salmonicida Triosephosphate isomerase (tpiA)

What is Triosephosphate isomerase (tpiA) and what is its role in bacterial metabolism?

Triosephosphate isomerase (EC 5.3.1.1) is a glycolytic enzyme that catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). This enzyme plays a critical role in central carbon metabolism by enabling efficient energy production during glycolysis. In bacterial systems like Aliivibrio salmonicida, tpiA is considered a housekeeping gene essential for basic cellular functions and energy production. The enzyme is particularly important for optimizing energy yield during glycolysis by ensuring that all carbon atoms from glucose can be utilized in the pathway .

Why is Aliivibrio salmonicida tpiA of interest to researchers?

Aliivibrio salmonicida is a psychrophilic (cold-loving) fish pathogen that causes cold-water vibriosis in marine fish. The study of its metabolic enzymes, including tpiA, provides insights into how these organisms adapt to cold environments. Research on A. salmonicida tpiA can help understand:

• Cold-adaptation mechanisms of enzymes

• Metabolic regulation in psychrophilic bacteria

• Potential virulence factors in fish pathogens

• Structure-function relationships in TIM barrel proteins

Similar to findings with tpiA in other pathogens like Salmonella, A. salmonicida tpiA may play a role in bacterial fitness and virulence, making it relevant for both basic research and applied studies in aquaculture disease management .

How does A. salmonicida tpiA differ from tpiA in mesophilic bacteria?

While specific comparative data for A. salmonicida tpiA is limited in the search results, psychrophilic enzymes typically exhibit distinctive adaptations compared to their mesophilic counterparts:

These adaptations allow psychrophilic enzymes to maintain flexibility and catalytic efficiency at lower temperatures, a critical requirement for A. salmonicida's survival in cold marine environments.

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

Based on experiences with other proteins from A. salmonicida, Escherichia coli-based expression systems can be used but may present challenges. For the ATP-dependent DNA ligase from A. salmonicida, toxicity to E. coli cells was a significant issue, which was overcome by fusion to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST) . A similar approach may be beneficial for tpiA expression:

• Recommended expression systems:

  • E. coli BL21(DE3) with pET or pBAD vectors for controlled expression

  • Fusion tag systems (MBP, GST, SUMO) to enhance solubility

  • Cold-shock promoters (cspA) that allow expression at lower temperatures (15-18°C)

  • Codon-optimized gene sequences to accommodate codon bias differences

• Expression conditions optimization:

  • Induction at lower temperatures (16-20°C) to promote proper folding

  • Reduced inducer concentrations

  • Extended expression times (overnight or longer)

  • Supplementation with osmolytes or chaperones

What purification strategies are most effective for recombinant A. salmonicida tpiA?

Drawing from the purification approach used for A. salmonicida ATP-dependent DNA ligase, a multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using the fusion tag (Ni-NTA for His-tagged proteins, amylose resin for MBP fusions, or glutathione sepharose for GST fusions)

• Nucleic acid removal: Treatment with a nuclease that can be inhibited by reducing agents (similar to the approach used for A. salmonicida DNA ligase)

• Tag removal: Site-specific protease cleavage (TEV, Factor Xa, or PreScission protease)

• Polishing steps:

  • Ion exchange chromatography

  • Size exclusion chromatography

  • Hydrophobic interaction chromatography

• Storage considerations:

  • Buffer optimization with glycerol (10-20%)

  • Temperature considerations (likely storage at -20°C or -80°C)

  • Avoiding repeated freeze-thaw cycles

How can researchers overcome common challenges in recombinant expression of A. salmonicida tpiA?

Common challenges and potential solutions include:

For A. salmonicida proteins with leader peptides, expressing both the full-length and mature forms (without the leader sequence) is advisable, as the truncated form may demonstrate higher activity, as observed with the ATP-dependent DNA ligase .

What methods are recommended for assessing the enzymatic activity of recombinant A. salmonicida tpiA?

Triosephosphate isomerase activity can be measured using several established methods:

• Coupled enzyme assay:

  • Measurement of NADH oxidation at 340 nm in a coupled system with α-glycerophosphate dehydrogenase

  • Reaction mixture typically contains tris-HCl buffer (pH 7.4-8.0), NADH, DHAP, and α-glycerophosphate dehydrogenase

• Direct assay:

  • Monitoring the conversion of G3P to DHAP by absorbance changes

  • Useful for determining kinetic parameters (Km, Vmax)

• Temperature-dependent activity profiles:

  • Activity measurements across a temperature range (0-40°C)

  • Critical for characterizing cold-adaptation properties

• pH-dependent activity profiles:

  • Activity measurements across pH range (6.0-9.0)

  • Important for understanding optimal conditions and environmental adaptations

For comprehensive characterization, researchers should assess both the activity and stability of the enzyme under various conditions relevant to the cold environments where A. salmonicida naturally exists.

How can researchers evaluate the structural features of A. salmonicida tpiA?

Structural characterization methods include:

• X-ray crystallography:

  • Gold standard for high-resolution protein structure determination

  • Requires high-purity protein and successful crystallization

  • Can reveal cold-adaptation structural features

• Circular dichroism (CD) spectroscopy:

  • For secondary structure content estimation

  • Thermal unfolding studies to determine stability

  • Comparison with mesophilic tpiA enzymes

• Differential scanning calorimetry (DSC):

  • Thermal stability analysis

  • Determination of melting temperature (Tm)

• Limited proteolysis:

  • Identification of flexible regions

  • Comparison with mesophilic counterparts

• Molecular dynamics simulations:

  • Computational analysis of flexibility and stability

  • Requires homology model or experimental structure

These methods together can provide insights into the structural basis of cold adaptation in A. salmonicida tpiA.

How can recombinant A. salmonicida tpiA be used to study cold adaptation mechanisms?

Recombinant A. salmonicida tpiA serves as an excellent model system for studying cold adaptation of enzymes:

• Comparative biochemistry:

  • Side-by-side activity assays with mesophilic and thermophilic tpiA

  • Activity and stability profiles across temperature ranges

  • Kinetic parameter determination at various temperatures

• Structure-function studies:

  • Site-directed mutagenesis of residues potentially involved in cold adaptation

  • Creation of chimeric enzymes with domains from mesophilic tpiA

  • Analysis of enzyme flexibility and catalytic efficiency at low temperatures

• Biophysical characterization:

  • Protein stability studies using thermal denaturation

  • Analysis of conformational dynamics using hydrogen-deuterium exchange

  • Fluorescence spectroscopy to monitor structural changes

• Computational approaches:

  • Molecular dynamics simulations at different temperatures

  • Computational prediction of flexibility and stability

  • Comparative sequence analysis across psychrophilic, mesophilic, and thermophilic organisms

What role might tpiA play in A. salmonicida pathogenesis and host adaptation?

Based on studies of tpiA in other pathogens, this enzyme may have significance beyond its metabolic role:

• Potential virulence factor:

  • In Salmonella enterica, tpiA deletion resulted in attenuated growth in a mouse model, suggesting its importance for in vivo fitness

  • Similar studies could be conducted with A. salmonicida in fish models

• Adaptation to host environment:

  • tpiA may be critical for adaptation to changing nutrient conditions within the host

  • Expression analysis during different stages of infection could reveal regulatory patterns

• Morphological changes:

  • tpiA deletion in Salmonella resulted in altered cell morphology (elongated shape)

  • Similar morphological impacts might occur in A. salmonicida and affect host interactions

• Vaccine development potential:

  • While tpiA deletion in Salmonella was not sufficiently attenuating for vaccine development , combination with other mutations might be explored for A. salmonicida

How can researchers utilize RT-qPCR to study tpiA expression patterns in A. salmonicida?

For accurate gene expression analysis of tpiA:

• Selection of appropriate reference genes:

  • For A. salmonicida, sdhA has been identified as a reliable housekeeping gene for RT-qPCR analysis, similar to Piscirickettsia salmonis

  • Using multiple validated reference genes is recommended for accurate normalization

• Experimental design considerations:

  • Growth conditions mimicking natural environment (temperature, salinity)

  • Different growth phases (lag, exponential, stationary)

  • Various stress conditions (temperature shifts, osmotic stress, nutrient limitation)

  • Infection models (in vitro or in vivo)

• Data analysis approach:

  • Normalization to total RNA rather than single housekeeping genes may provide more accurate results, especially when comparing tissues or treatments

  • Use of statistical methods to validate stability of reference genes

  • Application of proper quantification methods (ΔΔCt or standard curve)

What protein engineering approaches might enhance the utility of A. salmonicida tpiA for biotechnological applications?

Advanced protein engineering strategies include:

• Rational design:

  • Stability enhancement while maintaining low-temperature activity

  • Substrate specificity modification based on structural insights

  • Introduction of unnatural amino acids for specialized catalysis

• Directed evolution:

  • Error-prone PCR libraries screened for improved properties

  • DNA shuffling with other tpiA genes

  • Selection systems for enhanced activity or stability

• Computational design:

  • In silico prediction of beneficial mutations

  • Rosetta-based redesign of active site or interface regions

  • Machine learning approaches to identify non-obvious sequence-function relationships

• Enzyme immobilization:

  • Development of cold-active immobilized enzyme systems

  • Exploration of various carriers and immobilization techniques

  • Enhancement of operational stability at various temperatures

What is known about potential moonlighting functions of tpiA in bacterial systems?

Beyond its canonical role in glycolysis, tpiA may serve additional functions:

• Potential non-metabolic roles:

  • Involvement in bacterial adhesion or biofilm formation

  • Possible roles in stress response pathways

  • Interactions with host factors during infection

• Protein-protein interactions:

  • Identification of interaction partners through co-immunoprecipitation or yeast two-hybrid studies

  • Characterization of multi-protein complexes

  • Localization studies under different conditions

• Regulatory functions:

  • Potential roles in gene expression regulation

  • Involvement in sensing environmental conditions

  • Participation in feedback mechanisms

• Evolutionary considerations:

  • Analysis of selective pressures on tpiA sequence

  • Examination of conserved features beyond catalytic function

  • Comparative analysis across diverse bacterial species