Recombinant Gluconobacter oxydans ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in Gluconobacter oxydans metabolism?

ATP synthase subunit c (atpE) in G. oxydans is an essential component of the F1F0-ATP synthase complex, which is responsible for ATP production through oxidative phosphorylation. In G. oxydans, this protein plays a critical role in energy metabolism within this obligately aerobic organism. Unlike many other bacteria, G. oxydans has an incomplete oxidation pathway where it converts sugars and alcohols to corresponding aldehydes, ketones, and organic acids, which are then secreted into the medium . The ATP synthase complex helps maintain energy balance in this unique metabolic system.

The respiratory chain of G. oxydans is relatively simple compared to other organisms, but it contains numerous membrane-bound dehydrogenases that are crucial for the incomplete oxidation of substrates . The ATP synthase complex, including subunit c, works in conjunction with these dehydrogenases by utilizing the proton gradient generated by the respiratory chain for ATP synthesis. The subunit c forms a cylindrical oligomer that directly cooperates with subunit a in the proton pumping process .

How does the structure of ATP synthase subunit c in G. oxydans compare to other bacterial species?

While the search results don't provide specific structural information for G. oxydans ATP synthase subunit c, we can infer certain characteristics based on general ATP synthase knowledge and information from other organisms. In most bacteria, subunit c forms a cylindrical oligomer (typically c₁₀) that functions in proton translocation across the membrane .

What are the challenges in expressing recombinant G. oxydans ATP synthase subunit c?

Expressing recombinant membrane proteins like ATP synthase subunit c presents several methodological challenges:

• Membrane protein solubility: As an integral membrane protein, subunit c is highly hydrophobic and may aggregate when overexpressed, forming inclusion bodies that require solubilization and refolding.

• Proper folding and oligomerization: The functional form of subunit c requires proper assembly into the cylindrical oligomer, which may be difficult to achieve in heterologous expression systems.

• Host toxicity: Overexpression of membrane proteins often leads to toxicity in the host organism due to membrane stress.

• Post-translational modifications: Any required post-translational modifications must be properly executed by the host organism.

To address these challenges, researchers might consider using specialized expression strains designed for membrane proteins, fusion tags to improve solubility, and controlled expression conditions such as lower temperatures and optimized induction protocols. Expression in different hosts (E. coli, yeast, or homologous expression in G. oxydans itself) may yield different results depending on the specific research goals.

How does the ATP synthase subunit c contribute to the unique respiratory chain function in G. oxydans?

G. oxydans possesses a distinctive respiratory chain that supports its incomplete oxidation metabolism. Research indicates that G. oxydans contains both cytochrome bo₃-type oxidase (with high O₂ affinity) and cytochrome bd oxidase (with low O₂ affinity) . The ATP synthase complex, including subunit c, works in concert with these oxidases to maintain energy production under varying oxygen conditions.

The regulation of ATP synthase expression appears to be linked to oxygen sensing mechanisms in G. oxydans. The GoxR regulator (a fumarate-nitrate reduction regulator family member) functions as an oxygen sensor through an iron-sulfur cluster and regulates genes important for growth under oxygen-limited conditions . While GoxR directly regulates the cioAB genes encoding cytochrome bd oxidase and the pnt operon, its relationship with ATP synthase components including atpE would be an important area for advanced research.

Investigation of potential interactions between the ATP synthase complex and membrane-bound dehydrogenases would provide insights into how G. oxydans coordinates its energy metabolism with its industrial-relevant oxidation capabilities. Techniques such as protein-protein interaction studies, co-immunoprecipitation, and blue native PAGE could help elucidate these relationships.

What methods can be used to optimize recombinant expression of functional G. oxydans ATP synthase subunit c?

Optimizing recombinant expression of G. oxydans ATP synthase subunit c requires a multifaceted approach:

Table 1: Optimization Parameters for Recombinant G. oxydans ATP Synthase Subunit c Expression

For shake flask cultures, the choice of flask geometry can significantly impact protein production. Research on recombinant protein expression in Streptomyces has shown that baffled and coiled shake flasks produce smaller mycelial clumps compared to conventional flasks, resulting in approximately 3 times higher recombinant protein production . Similar principles may apply to G. oxydans cultures, where proper mixing and oxygenation are critical.

Advanced researchers should consider resonant acoustic mixing (RAM) as an alternative to orbital mixing (OM) for shake flask cultures. Studies with recombinant E. coli have demonstrated that RAM can increase biomass yield by ~69% compared to OM at the same initial volumetric oxygen transfer coefficient . This technique could potentially improve the yield of recombinant ATP synthase subunit c by enhancing oxygen transfer and reducing stress on the cells.

How do post-translational modifications affect G. oxydans ATP synthase subunit c function?

While specific information on post-translational modifications (PTMs) of G. oxydans ATP synthase subunit c is not directly available in the search results, this represents an important research question. In other organisms, ATP synthase subunits can undergo various PTMs including phosphorylation, acetylation, and other modifications that affect their function.

For G. oxydans, which grows optimally in acidic environments and has adapted to rapid incomplete oxidation of substrates, PTMs might play a role in regulating ATP synthase activity under different environmental conditions. Research methodologies to investigate this could include:

• Mass spectrometry analysis of native ATP synthase subunit c isolated from G. oxydans grown under different conditions

• Site-directed mutagenesis of potential modification sites in recombinant constructs

• In vitro modification assays using purified kinases, acetylases, or other modification enzymes

• Comparative proteomic analysis between G. oxydans and related bacteria with different metabolic capabilities

Understanding these modifications could provide insights into how G. oxydans regulates energy production in response to environmental changes and substrate availability.

What expression systems are most suitable for producing recombinant G. oxydans ATP synthase subunit c?

The choice of expression system for recombinant G. oxydans ATP synthase subunit c depends on research objectives:

For structural studies:
E. coli C41/C43(DE3) strains, specifically designed for membrane protein expression, offer high yields. These strains have mutations that prevent the toxicity associated with membrane protein overexpression. Codon optimization of the G. oxydans atpE gene for E. coli expression would improve translation efficiency.

For functional studies:
Homologous expression in G. oxydans itself may preserve native interactions and post-translational modifications. The G. oxydans strain 621H, whose genome has been fully sequenced , would be an appropriate host. This approach would require development of inducible expression vectors specific for G. oxydans.

For interaction studies:
Yeast expression systems like Pichia pastoris may provide a eukaryotic-like membrane environment while maintaining high expression levels. Addition of appropriate antifoam agents to shake flask cultures of P. pastoris has been shown to increase recombinant protein yield , which could be beneficial for ATP synthase subunit c production.

Each system requires optimization of culture conditions, including temperature, pH, oxygen levels, and induction parameters. For membrane proteins like ATP synthase subunit c, lower induction temperatures (16-25°C) often improve proper folding and reduce inclusion body formation.

How can I design effective mutagenesis studies for G. oxydans ATP synthase subunit c?

Designing effective mutagenesis studies for G. oxydans ATP synthase subunit c requires:

• Structure-based targeting: Use homology modeling based on known bacterial ATP synthase structures to identify critical residues. Focus on:

  • Proton-binding sites in the transmembrane domain

  • Oligomerization interfaces

  • Interaction surfaces with other ATP synthase subunits

• Conservation analysis: Compare the atpE sequence from G. oxydans with other bacteria, particularly those with similar metabolic characteristics. Conserved residues often indicate functional importance, while divergent residues may reflect adaptations specific to G. oxydans' unique metabolism.

• Systematic mutagenesis approaches:

  • Alanine scanning of transmembrane domains

  • Charge-altering mutations at potential proton-binding sites

  • Conservative vs. non-conservative substitutions of key residues

  • Domain swapping with other bacterial ATP synthase c subunits

• Phenotypic assessment methods:

  • Growth rate analysis under various conditions

  • ATP synthesis activity measurements

  • Proton pumping assays

  • Protein-protein interaction studies

When expressing mutants, consider using the native G. oxydans promoter and regulatory elements to maintain physiologically relevant expression levels. This approach has been successful in studying the GoxR regulator in G. oxydans .

What techniques can be used to study the assembly of recombinant ATP synthase subunit c into functional oligomers?

Studying the assembly of recombinant ATP synthase subunit c into functional oligomers requires specialized techniques:

• Blue Native PAGE: This technique separates protein complexes in their native state and can verify the formation of c-ring oligomers. Coupled with second-dimension SDS-PAGE, it can identify interaction partners.

• Cross-linking mass spectrometry: Chemical cross-linking followed by mass spectrometry analysis can identify proximity relationships between subunits and determine oligomeric arrangements.

• Förster Resonance Energy Transfer (FRET): By tagging subunit c with appropriate fluorophores, FRET can measure distances between subunits and monitor oligomerization dynamics in real-time.

• Analytical ultracentrifugation: This technique can determine the stoichiometry of the c-ring oligomer in G. oxydans, which may differ from the typical c₁₀ arrangement found in many bacteria.

• Cryo-electron microscopy: For high-resolution structural analysis of the assembled c-ring and its interactions with other ATP synthase components.

When expressing recombinant ATP synthase subunit c for these studies, controlling the culture conditions is critical. Research on other recombinant systems has shown that parameters such as flask geometry and mixing method can significantly affect protein quality . For instance, cultures subjected to resonant acoustic mixing have shown differences in protein aggregation patterns compared to orbital mixing, which could impact the proper assembly of oligomeric membrane proteins like ATP synthase subunit c.

How can I differentiate between direct and indirect effects when analyzing mutants of G. oxydans ATP synthase subunit c?

Differentiating between direct and indirect effects in ATP synthase subunit c mutants requires a comprehensive analytical approach:

• Combine structural and functional analyses:

  • Structural: Assess proper folding and oligomerization of mutant subunit c using techniques like circular dichroism spectroscopy and native PAGE

  • Functional: Measure ATP synthesis rates and proton translocation in reconstituted systems

• Perform epistasis analysis:

  • Create double mutants combining ATP synthase subunit c mutations with mutations in related systems (e.g., respiratory chain components)

  • Analyze suppressor mutations that restore function in defective mutants

• Use controlled expression systems:

  • Implement titratable expression systems to normalize protein levels across mutants

  • Account for potential differences in protein stability with pulse-chase experiments

• Apply systems biology approaches:

  • Transcriptomics to identify compensatory changes in gene expression

  • Metabolomics to detect shifts in metabolic pathways that might mask or amplify primary phenotypes

• Establish clear control experiments:

  • Include wild-type controls grown under identical conditions

  • Use active-site mutants as negative controls for functional assays

  • Include unrelated membrane protein mutants to distinguish general membrane effects from ATP synthase-specific effects

The GoxR regulator study in G. oxydans provides a methodological template, where researchers combined transcriptome comparisons between mutant and wild-type strains with in vivo binding site detection (ChAP-Seq) to identify direct targets . Similar approaches could be adapted for ATP synthase subunit c studies.

What standards should be used to evaluate the quality of recombinant G. oxydans ATP synthase subunit c preparations?

Evaluating recombinant G. oxydans ATP synthase subunit c quality requires multiple quality control checkpoints:

Table 2: Quality Standards for Recombinant ATP Synthase Subunit c Preparations

When expressed in inclusion bodies, analysis of secondary structure content using ATR-FTIR can provide valuable insights into protein quality. Research on recombinant proteins in E. coli has shown that culture conditions can affect the proportion of α-helices and β-sheets in inclusion bodies , which could impact later refolding and reconstitution steps for ATP synthase subunit c.

For membrane-integrated preparations, fluorescence recovery after photobleaching (FRAP) can assess protein mobility within the membrane, which correlates with proper folding and integration. Cryo-electron microscopy of reconstituted proteoliposomes can confirm proper insertion and oligomerization.

How can I interpret conflicting results between in vitro and in vivo studies of ATP synthase subunit c function?

Interpreting conflicting results between in vitro and in vivo studies requires systematic troubleshooting:

• Examine differences in experimental context:

  • In vitro studies may lack essential interaction partners or regulatory factors

  • In vivo studies are affected by cellular homeostasis mechanisms that may mask primary effects

• Consider the membrane environment:

  • The lipid composition can significantly affect ATP synthase function

  • In vitro reconstitution may not replicate the native membrane environment of G. oxydans

  • Acidophilic bacteria like G. oxydans may have specific membrane adaptations

• Assess protein modifications and processing:

  • In vivo post-translational modifications may be absent in recombinant systems

  • The targeting peptide of ATP synthase subunit c may have additional functions beyond import, as demonstrated for mammalian ATP synthase subunit c isoforms

• Evaluate concentration and stoichiometry effects:

  • Non-physiological protein concentrations in vitro may alter equilibrium or kinetics

  • Improper stoichiometry of ATP synthase components in reconstituted systems

• Design bridging experiments:

  • Perform in vitro assays with increasing complexity (purified protein → membrane extracts → permeabilized cells)

  • Use genetic approaches to create controlled in vivo systems (depletion strains, conditional mutants)

Research on mammalian ATP synthase subunit c has shown that silencing individual isoforms affects not only ATP synthesis but also respiratory chain structure and function . This suggests complex interrelationships that may not be fully captured in simplified in vitro systems, highlighting the importance of combining multiple experimental approaches when studying G. oxydans ATP synthase subunit c.

How might the unique metabolism of G. oxydans influence ATP synthase subunit c evolution and function?

G. oxydans has evolved a specialized metabolism focused on incomplete oxidation of substrates, which may have driven adaptations in its energy conservation systems. Research into ATP synthase subunit c evolution could explore:

• Comparative genomics approach: Analysis of ATP synthase subunit c sequences across acetic acid bacteria with different metabolic capabilities could reveal adaptation signatures. G. oxydans' genome has been fully sequenced , facilitating such comparisons.

• Structure-function relationship: The unique metabolic environment of G. oxydans, including operation at low pH and high sugar concentrations , may have selected for specific structural features in ATP synthase subunit c. Homology modeling coupled with molecular dynamics simulations could identify these adaptations.

• Regulatory network analysis: The connection between oxygen-sensing mechanisms (like GoxR ) and ATP synthase regulation could provide insights into how G. oxydans coordinates energy production with its incomplete oxidation metabolism.

• Metabolic flux analysis: Quantifying how ATP synthase activity responds to different carbon sources and oxygen levels could reveal metabolic integration mechanisms specific to G. oxydans.

These studies would contribute to our understanding of how essential cellular machinery like ATP synthase adapts to support specialized metabolic capabilities, with potential applications in metabolic engineering for biotechnological applications.

What role might ATP synthase subunit c play in G. oxydans adaptation to industrial conditions?

G. oxydans is industrially valuable for its incomplete oxidation capabilities, particularly in vitamin C production . Understanding how ATP synthase subunit c contributes to industrial robustness could involve:

• Stress response studies: Investigate how industrial stressors (high substrate concentrations, oxidative stress, pH fluctuations) affect ATP synthase expression and assembly.

• Engineered variants: Design and test ATP synthase subunit c variants with enhanced stability under industrial conditions using directed evolution approaches.

• Energy efficiency analysis: Quantify how ATP synthase efficiency correlates with industrial productivity metrics. More efficient energy conservation might improve biomass yield, which is typically low in G. oxydans due to its incomplete oxidation metabolism .

• Comparative performance in different bioreactor configurations: Evaluate ATP synthase function and expression in various cultivation systems, from shake flasks to industrial bioreactors. Research has shown that scaling up from shake flasks to bioreactors requires careful consideration of parameters like power input and morphology .

These investigations could inform strain improvement strategies for industrial applications while providing fundamental insights into bioenergetic adaptations in specialized metabolism.