Recombinant Aliivibrio salmonicida ATP synthase subunit c (atpE)

What is the molecular structure and characteristics of Aliivibrio salmonicida ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) from Aliivibrio salmonicida is a small membrane protein (85 amino acids) that forms part of the F0 sector of the F-type ATP synthase complex. The protein has the following characteristics:

• Full amino acid sequence: METLLSFSAIAVGIIVGLASLGTAIGFAILGGKFLEGAARQPEMAPMLQVKMFIIAGLLDAVPMIGIVIALLFTFANPFVGQLAG

• UniProt ID: B6EHU2

• Gene locus: VSAL_I3064

• Alternative names: ATP synthase F(0) sector subunit c, F-type ATPase subunit c, Lipid-binding protein

The protein contains transmembrane α-helices involved in proton translocation across the membrane as part of ATP synthesis. Unlike many bacterial homologs with a GxGxGxG motif, some extremophiles have adapted this region with alternative amino acid patterns to optimize function in challenging environments .

How should recombinant Aliivibrio salmonicida ATP synthase subunit c (atpE) be stored and handled for optimal stability?

For optimal stability and activity maintenance of recombinant Aliivibrio salmonicida ATP synthase subunit c:

• Storage temperature: Store at -20°C for regular use or -80°C for extended storage

• Buffer composition: Typically maintained in Tris-based buffer with 50% glycerol or Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Handling recommendations:

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • Centrifuge vials briefly before opening to bring contents to the bottom

  • For reconstitution of lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Consider adding 5-50% glycerol (final concentration) for long-term storage

What expression systems are most suitable for producing recombinant Aliivibrio salmonicida ATP synthase subunit c?

Based on current research protocols, the optimal expression system for recombinant Aliivibrio salmonicida ATP synthase subunit c is:

• Host organism: E. coli is the preferred expression system

• Vector design: Vectors containing an N-terminal His-tag facilitate purification

• Expression region: Full-length protein (amino acids 1-85)

• Protein tag options: His-tag is commonly used, though the tag type may be determined during the production process depending on experimental needs

The use of E. coli allows for high yield production while maintaining proper folding of this relatively small membrane protein. For membrane proteins like atpE, expression conditions typically require optimization of induction temperature, inducer concentration, and incubation time to balance between protein yield and proper folding .

What methodological approaches are recommended for investigating interactions between ATP synthase subunit c and other components of the ATP synthase complex?

To study the interactions between ATP synthase subunit c and other complex components, researchers should consider the following methodological approaches:

Table 1: Recommended methods for studying atpE protein interactions

When conducting these experiments, it's crucial to consider the membrane environment, as ATP synthase subunit c functions within the lipid bilayer. Using detergents that mimic the native membrane environment while still allowing solubilization is essential for maintaining physiologically relevant interactions .

How can researchers optimize experimental design for studying the role of ATP synthase subunit c in bacterial energy metabolism?

When designing experiments to investigate ATP synthase subunit c function in energy metabolism:

• ATP synthesis measurement: Use luciferin-luciferase assays with digitonin-permeabilized cells using substrates like malate plus pyruvate to measure ATP production capacity .

• Oxygen consumption analysis: Employ Clark-type electrodes to measure respiratory chain function in intact cells, with inhibitors like KCN to confirm mitochondrial respiration dependence .

• Membrane potential assessment: Use fluorescent dyes such as TMRM to evaluate membrane potential maintenance, which is crucial for ATP synthase function .

• Gene silencing approach: For functional studies, RNA interference with siRNAs targeting specific regions of the atpE transcript can reveal the protein's role. Include appropriate controls with scrambled oligonucleotides .

• Growth condition variations: Test bacterial growth under limiting energy sources at different pH values to assess the impact of ATP synthase function on cell physiology. For example, when studying extremophiles, compare growth at both neutral pH and extreme conditions (e.g., pH >10 for alkaliphiles) .

Data analysis should include comparison of ATP synthesis rates, oxygen consumption, and growth rates between wildtype and manipulated cells, with statistical analysis to determine significance of observed differences .

What techniques are most effective for investigating the stoichiometry and structure of ATP synthase c-rings in Aliivibrio salmonicida?

The c-ring stoichiometry is critical for ATP synthase function as it determines the ion-to-ATP ratio. For investigating c-ring structure and stoichiometry:

Recommended techniques include:

• Atomic Force Microscopy (AFM): Provides single-protein complex resolution to directly visualize c-ring structure and count subunits. This technique has been successfully used to determine c-ring stoichiometry in other bacterial species .

• X-ray Crystallography: Offers high-resolution structural data at the c/c-subunit contacting interface. This provides detailed information about the molecular arrangements within the c-ring .

• Cryo-electron Microscopy: Allows visualization of the complete ATP synthase complex including the c-ring in a near-native state without crystallization.

• Mass Spectrometry: Can be used to determine the precise molecular weight of intact c-rings, which can be used to calculate stoichiometry.

When interpreting results, researchers should consider that:

• C-ring stoichiometry can vary between species (typically 10-15 subunits)

• Environmental adaptations may affect the optimal c-ring size

• The ion-to-ATP ratio (i = cn/β3) is directly impacted by c-ring stoichiometry and affects cellular bioenergetics

How can researchers optimize qPCR experimental design when studying atpE gene expression in Aliivibrio salmonicida?

For optimal qPCR analysis of atpE gene expression in Aliivibrio salmonicida:

• Experimental design optimization:

  • Use a dilution-replicate design instead of identical replicates

  • Perform a single reaction at several dilutions for every test sample

  • This approach allows simultaneous measurement of PCR efficiency and quantity

• Efficiency determination:

  • For dilution-replicate design, use the formula: T = Q(Cq) = Q(0) × E^Cq × d

  • All standard curves can be simultaneously fit with the constraint of slope equality

  • This results in a globally estimated efficiency (E)

• Control considerations:

  • Include reference genes that maintain stable expression under your experimental conditions

  • For multi-run experiments, include at least one identical sample in each run to control for inter-run variation

• Data analysis approach:

  • Plot log(dilution) vs Cq values to obtain a linear relationship

  • For n dilution points across m samples, the number of degrees of freedom for estimating global efficiency is [(dilution points - 1) × (number of samples) - 1]

This optimized approach reduces the number of required reactions while maintaining or improving the precision of results compared to traditional qPCR methods .

What approaches should be used to resolve discrepancies in experimental results when studying Aliivibrio salmonicida ATP synthase subunit c?

When encountering conflicting data in atpE research, implement this systematic approach:

• Systematically exploit inconsistencies: Use contradictions between model predictions and experimental results as opportunities to improve methodological approaches .

• Reconciliation protocol:

  • Categorize inconsistencies (e.g., expression levels, functional assays, interaction studies)

  • For each category, identify potential sources of variation in experimental methods

  • Design targeted experiments to specifically address the source of discrepancy

• Model refinement:

  • When experimental results contradict computational predictions, use these differences to improve computational models

  • Implement iterative refinement by using experimental data to update model parameters

• Experimental validation approaches:

  • Employ complementary techniques to verify results (e.g., if Western blot and qPCR data conflict, add proteomics analysis)

  • Use genetic complementation to confirm phenotypes (expression of wild-type atpE should rescue knockout phenotypes)

  • Consider environmental variables like temperature, pH, and salt concentration that might affect protein function differently in Aliivibrio salmonicida compared to model organisms

This systematic approach has been shown to resolve up to 45% of inconsistencies in similar biological model systems .

How can researchers investigate the role of ATP synthase subunit c in Aliivibrio salmonicida pathogenicity and cold adaptation?

To explore the connection between ATP synthase subunit c and A. salmonicida pathogenicity:

• Infection model development:

  • Utilize Atlantic salmon (Salmo salar) experimental challenge models

  • Compare virulence of wild-type bacteria versus atpE mutants

  • Monitor development of cold-water vibriosis symptoms and mortality rates

• Temperature-dependent studies:

  • Examine ATP synthase activity at different temperatures (4°C to 20°C)

  • Investigate whether atpE expression is temperature-regulated

  • Compare ATP synthase complex stability at different temperatures

• Integration with quorum sensing systems:

  • Explore potential interaction between ATP synthase function and the LuxI-LuxR or AinS-AinR quorum sensing systems

  • Analyze whether ATP availability affects virulence factor expression

  • Test for co-regulation of atpE with known virulence determinants

• Biofilm formation analysis:

  • Evaluate the role of ATP synthase in biofilm development using crystal violet staining and confocal microscopy

  • Compare biofilm formation between wild-type and atpE-modified strains

  • Assess whether ATP limitation alters biofilm structures at cold temperatures

When interpreting results, consider that A. salmonicida's pathogenicity mechanisms are complex and may involve connections between energy metabolism, temperature adaptation, and virulence systems that are not present in model organisms .

What are the most effective mutagenesis approaches for studying structure-function relationships in Aliivibrio salmonicida ATP synthase subunit c?

When designing mutation studies of atpE to understand structure-function relationships:

• Target selection strategies:

  • Focus on the transmembrane α-helix packing motif (equivalent to GxGxGxG in other bacteria)

  • Target the ion-binding site residues involved in proton translocation

  • Examine residues at the c-c subunit interface that may affect c-ring assembly

• Mutagenesis approaches:

  • Site-directed mutagenesis for precise amino acid substitutions

  • Alanine-scanning mutagenesis to systematically evaluate residue contributions

  • Conservative vs. non-conservative substitutions to assess chemical property requirements

• Functional assessment protocol:

  • Measure ATP synthesis capacity in mutants using luciferin-luciferase assays

  • Evaluate proton translocation efficiency using pH-sensitive fluorescent dyes

  • Assess c-ring assembly and stability through blue native PAGE or AFM

• Complementation testing:

  • Express wild-type or mutant atpE genes in knockout strains

  • Test cross-complementation between different variants to identify functional domains

  • Use targeting peptide swap experiments to investigate domain-specific functions

Table 2: Key residues for mutagenesis in ATP synthase subunit c

Results from such mutations should be interpreted in the context of A. salmonicida's adaptation to its environmental niche, as sequence variations from model organisms may reflect specific adaptations rather than merely structural requirements .