Recombinant Oenococcus oeni ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c (atpE) in Oenococcus oeni metabolism?

ATP synthase subunit c (atpE) in O. oeni forms part of the F0 domain of the F0F1-ATP synthase complex, which is essential for energy transduction. This protein functions as part of the proton-conducting channel that harnesses the proton motive force to generate ATP. In O. oeni, ATP synthase activity has been directly associated with malolactic fermentation (MLF), playing a crucial role in the bacterium's survival under stress conditions .

Research has demonstrated that ATP synthase contributes approximately 45-48% of the total ATP production in O. oeni cells, with the remainder coming from heterolactic fermentation pathways . This balance shifts slightly as environmental conditions change, particularly in response to ethanol stress, which is significant for the bacterium's survival in wine environments.

How does ATP synthase expression change under wine-related stress conditions?

Transcriptomic analyses reveal that several ATP synthase subunit genes, including those coding for β, γ, and ε subunits, are significantly up-regulated after acid shock at pH 3.0 compared to control conditions (pH 4.8) . The following table summarizes the expression patterns observed:

This differential expression pattern suggests that ATP synthase activity is rapidly modulated as part of the acid stress response mechanism, which differs from the response pattern observed in wine-like medium .

How does atpE contribute to proton extrusion in acidic environments?

The atpE gene encodes the c subunit that forms the c-ring in the F0 domain of ATP synthase. Under acidic conditions, O. oeni cells must maintain cytoplasmic pH homeostasis by extruding protons. Research indicates that at 12% ethanol concentration, O. oeni spends approximately 30 times more ATP to maintain viability than in the absence of ethanol, with most of this ATP consumed for proton extrusion .

The c subunit is integral to this process as it contains the proton-binding site (typically a conserved carboxylate residue) that facilitates proton translocation across the membrane. Experimental evidence suggests that the proton-pumping activity mediated by ATP synthase is a critical determinant of O. oeni's ability to survive in acidic wine conditions.

What protocols yield optimal expression of recombinant O. oeni atpE in heterologous systems?

Successful expression of recombinant O. oeni atpE requires careful optimization of several parameters. Based on protocols developed for similar membrane proteins, the following methodology is recommended:

• Vector Selection: pET expression systems with a hexahistidine tag facilitate purification.

• Host Strain: E. coli C41(DE3) or C43(DE3) strains, which are specialized for membrane protein expression.

• Expression Conditions:

  • Induction with 0.5 mM IPTG at OD600 of 0.6-0.8

  • Post-induction growth at 18-20°C for 16-18 hours to minimize inclusion body formation

  • Supplementation with 1% glucose to repress basal expression

When expressing hydrophobic membrane proteins like atpE, inclusion of detergents (0.2-0.5% n-dodecyl-β-D-maltoside) in the lysis buffer is essential for solubilization. Purification typically employs immobilized metal affinity chromatography followed by size exclusion chromatography to obtain homogeneous protein preparations.

How does the ATP synthase activity correlate with ethanol tolerance in O. oeni?

Experimental data indicate a complex relationship between ATP synthase activity and ethanol tolerance in O. oeni. As shown in the following figure based on data from search result :

Researchers investigating this phenomenon should employ a combination of biochemical assays measuring ATP synthesis rates and molecular techniques to assess gene expression under varying ethanol concentrations.

What experimental approaches can determine the stoichiometry of the c-ring in O. oeni ATP synthase?

Determining the stoichiometry of the c-ring is crucial for understanding the bioenergetic efficiency of ATP synthesis. The following methods are recommended:

• Atomic Force Microscopy (AFM): Provides direct visualization of the c-ring structure after isolation and reconstitution into lipid bilayers.

• Mass Spectrometry of Intact Complexes: Electrospray ionization mass spectrometry can determine the mass of the entire c-ring, from which the number of c subunits can be calculated.

• Cross-linking Studies: Chemical cross-linking coupled with SDS-PAGE analysis can reveal the oligomeric state of the c-ring.

• Cryo-Electron Microscopy: Provides structural information that can determine the number of c subunits in the assembled ring.

The stoichiometry directly impacts the H+/ATP ratio, which in turn affects the bioenergetic efficiency of ATP synthesis. The experimental design should include appropriate controls and validation using multiple complementary techniques.

How can researchers design experiments to elucidate the interaction between atpE and other ATP synthase subunits in O. oeni?

Investigating subunit interactions within the ATP synthase complex requires a multifaceted experimental approach:

• Bacterial Two-Hybrid Assays: Modified for membrane proteins to identify potential interaction partners of atpE.

• Co-immunoprecipitation Studies: Using antibodies against atpE or tagged versions of the protein to isolate interacting partners.

• FRET Analysis: Employing fluorescently labeled subunits to detect proximity and interaction in vivo.

• Cross-linking Mass Spectrometry: Identifying interaction interfaces by cross-linking followed by mass spectrometric analysis.

The experimental design should follow these steps:
a. Generate constructs expressing atpE with different tags
b. Validate expression and functionality
c. Perform interaction assays under native and stress conditions
d. Analyze data using appropriate statistical methods
e. Validate interactions using orthogonal methods

When investigating membrane protein interactions, it is essential to maintain the native membrane environment or use appropriate detergents that preserve the structural integrity of the complex.

What are the methodological considerations for analyzing the impact of atpE mutations on O. oeni stress resistance?

A comprehensive analysis of atpE mutations requires:

• Site-Directed Mutagenesis: Target conserved residues in atpE, particularly proton-binding sites and residues at subunit interfaces.

• Phenotypic Characterization: Assess growth under various stress conditions:

  • Acidic pH (3.0-4.5)

  • Ethanol stress (8-14%)

  • Temperature stress (15-25°C)

  • Combined stressors to mimic wine conditions

• Enzymatic Activity Measurements: Determine ATP synthesis rates and proton pumping activities of mutant strains.

• Transcriptional Response Analysis: Use RNA-Seq to profile global transcriptional changes in mutant strains compared to wild-type.

The experimental design should include at least three biological replicates and appropriate statistical analysis (ANOVA with post-hoc tests) to ensure robustness. Control experiments should include complementation with wild-type atpE to confirm the phenotypic effects are due to the introduced mutations.

According to transcriptomic data, acid stress in O. oeni triggers differential expression of multiple stress response genes alongside ATP synthase components . Therefore, researchers should evaluate both direct effects on ATP synthase function and indirect effects on general stress response pathways.

How can advanced biophysical techniques be applied to study the proton translocation mechanism through the O. oeni atpE c-ring?

Understanding proton translocation through the c-ring requires sophisticated biophysical approaches:

• Solid-State NMR Spectroscopy: Provides atomic-level insights into the protonation/deprotonation dynamics of key residues in the c-ring.

• Single-Molecule FRET: Monitors conformational changes associated with proton binding and release.

• Electrophysiological Measurements: Using reconstituted proteoliposomes to measure proton currents across membranes containing purified c-rings.

• Molecular Dynamics Simulations: Model proton movement through the c-ring based on structural data.

The experimental workflow should include:
a. Expression and purification of the c-ring
b. Reconstitution into appropriate membrane mimetics
c. Biophysical measurements under varying pH and ionic conditions
d. Integration of experimental data with computational models

These studies should be performed at physiologically relevant pH values (pH 3.0-4.5) to simulate wine conditions where O. oeni typically functions. Special attention should be paid to the influence of ethanol on membrane properties and protein dynamics, as ethanol concentrations in wine significantly impact ATP synthase function .

How can researchers overcome challenges in obtaining functional recombinant O. oeni atpE?

Common challenges in recombinant atpE production include:

• Protein Aggregation: Optimize by:

  • Reducing expression temperature to 16°C

  • Using specialized strains like E. coli Lemo21(DE3)

  • Adding stabilizing agents (glycerol, specific lipids) to buffers

• Low Expression Yields: Enhance through:

  • Codon optimization for the expression host

  • Using stronger promoters or inducible systems

  • Testing multiple fusion tags (MBP, SUMO) to increase solubility

• Loss of Function During Purification: Preserve activity by:

  • Maintaining constant pH throughout purification

  • Including appropriate lipids in purification buffers

  • Using mild detergents (DDM, LMNG) for solubilization

• Proper Folding Verification: Assess using:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Limited proteolysis to verify native conformation

  • Activity assays to confirm functionality

Each modification should be systematically tested and documented to establish optimal conditions for your specific experimental system.

What statistical approaches are most appropriate for analyzing ATP synthase activity data from O. oeni under multiple stress conditions?

When analyzing complex datasets involving multiple variables (pH, ethanol content, temperature) affecting ATP synthase activity:

• Multifactorial ANOVA: Essential for determining main effects and interactions between different stressors.

• Response Surface Methodology (RSM): Provides a mathematical model describing how multiple factors affect ATP synthase activity simultaneously.

• Principal Component Analysis (PCA): Useful for identifying patterns and reducing dimensionality in large datasets.

• Linear Mixed Effects Models: Appropriate when incorporating both fixed effects (experimental variables) and random effects (biological variation).

Researchers should:
a. Ensure appropriate sample sizes (power analysis before experiments)
b. Test for normality and homogeneity of variance
c. Consider appropriate transformations if assumptions are violated
d. Include relevant covariates in the analysis
e. Use post-hoc tests with appropriate corrections for multiple comparisons

RNA-seq data analysis for transcriptional responses should employ specialized statistical frameworks like DESeq2 or edgeR to account for the unique characteristics of count data .

How might genome editing technologies be applied to study atpE function in O. oeni?

CRISPR-Cas9 and related technologies offer promising approaches for precise genetic manipulation of O. oeni, which has traditionally been challenging to modify genetically:

• Development of Specific Protocols:

  • Optimize transformation conditions for O. oeni

  • Design sgRNAs targeting atpE with minimal off-target effects

  • Develop efficient homology-directed repair templates

• Potential Applications:

  • Generate point mutations in conserved residues of atpE

  • Create conditional knockdown strains

  • Introduce reporter fusions to monitor expression dynamics

• Methodological Considerations:

  • Use inducible CRISPR systems to control editing timing

  • Employ counter-selection markers for efficient isolate screening

  • Validate edits through sequencing and functional assays

The implementation of genome editing will enable precise correlation between specific amino acid residues in atpE and functional outcomes related to ATP synthesis, proton pumping, and stress tolerance.

What integrated omics approaches would provide comprehensive insights into ATP synthase function in O. oeni?

A multi-omics strategy would yield the most complete understanding of ATP synthase function in the context of O. oeni metabolism:

• Genomics: Compare atpE sequences across multiple O. oeni strains to identify conserved and variable regions.

• Transcriptomics: Analyze expression patterns of ATP synthase genes under different conditions, as demonstrated in existing research showing differential expression under acid stress .

• Proteomics: Quantify ATP synthase subunit abundance and post-translational modifications.

• Metabolomics: Track ATP/ADP ratios and related metabolites under varying conditions.

• Fluxomics: Measure metabolic flux distributions to quantify ATP production rates.

Integration of these datasets requires:
a. Synchronized experimental designs
b. Consistent sample preparation
c. Appropriate normalization methods
d. Advanced computational tools for data integration
e. Validation experiments to confirm predictions

This approach would provide unprecedented insights into how ATP synthase activity coordinates with broader metabolic networks during malolactic fermentation and under stress conditions.