Recombinant Shewanella sp. ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Shewanella species?

ATP synthase subunit c in Shewanella oneidensis is a small, hydrophobic protein comprising 83 amino acids, forming the membrane-embedded c-ring of the F₀ portion of ATP synthase. The amino acid sequence (METILGMTAIAVALLIGMGALGTAIGFGLLGGKFLEGAARQPEMAPMLQVKMFIVAGLLDAVTMIGVGIALFMLFTNP) reveals its highly hydrophobic nature, consistent with its role in the membrane domain . This protein facilitates proton translocation across the membrane during ATP synthesis, contributing to the chemiosmotic mechanism that powers ATP formation.

The c-subunit is part of the proton channel and participates in the rotary mechanism that couples proton flow to ATP synthesis. In Shewanella species, this process is particularly important given their metabolically versatile lifestyle and ability to thrive in diverse environments using various electron acceptors.

How does ATP synthase contribute to Shewanella's energy metabolism under different respiratory conditions?

Shewanella oneidensis MR-1 is a facultative anaerobe with remarkable respiratory versatility. Research indicates that the proportion of ATP produced through oxidative phosphorylation (involving ATP synthase) versus substrate-level phosphorylation varies significantly depending on electron acceptor availability. Under oxygen-limited conditions with lactate as carbon source, approximately 33% of ATP comes from substrate-level phosphorylation, while the remainder must be generated through ATP synthase activity .

When growing anaerobically with fumarate as the electron acceptor, the contribution of substrate-level phosphorylation increases to as much as 72.5% of the total ATP required for growth . This suggests that ATP synthase activity is modulated based on respiratory conditions. Interestingly, under certain anaerobic conditions, the Pta-AckA pathway for substrate-level phosphorylation becomes essential, while F₀F₁ ATP synthase function becomes less critical, as evidenced by minimal growth defects in ATP synthase mutants .

What is known about atpE gene regulation in Shewanella species?

While the search results don't provide direct information about atpE regulation, we can infer from Shewanella's metabolic flexibility that ATP synthase expression is likely responsive to electron acceptor availability and energy demands. The organism's ability to shift between aerobic respiration, anaerobic respiration with diverse electron acceptors, and even pyruvate fermentation suggests sophisticated regulatory mechanisms for energy metabolism genes.

How can genetic code expansion be applied to study ATP synthase function in Shewanella?

Recent advances in genetic code expansion techniques for Shewanella oneidensis MR-1 provide powerful tools for investigating ATP synthase function. Researchers have successfully developed methods for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins expressed in S. oneidensis MR-1 using the Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNA (MbPylRS/tRNACUA) pair and the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjCNFRS)/tRNACUA pair .

This technology could be applied to incorporate ncAAs with specialized chemical properties into ATP synthase subunit c to:

• Introduce photo-crosslinking amino acids to capture transient protein-protein interactions within the ATP synthase complex

• Incorporate bioorthogonal functional groups for site-specific fluorescent labeling to visualize ATP synthase localization and dynamics

• Add environmentally sensitive probes to monitor conformational changes during the catalytic cycle

The research has demonstrated that this approach is compatible with S. oneidensis protein synthesis, maturation, and secretion pathways , suggesting it could be effectively applied to membrane proteins like ATP synthase components.

What role might ATP synthase play in the metabolic engineering of Shewanella for bioproduction?

Shewanella oneidensis has emerged as a promising platform for bioproduction, particularly for compounds derived from central carbon metabolism. Recent metabolic engineering efforts have focused on optimizing glutamate production and enabling itaconic acid synthesis by redirecting carbon flux towards the TCA cycle .

ATP synthase likely plays a critical role in these engineered pathways by:

• Influencing the energetics of engineered production pathways

• Maintaining redox balance during product formation

• Responding to altered electron flow in redirected metabolic pathways

For example, when S. oneidensis was engineered for glutamate production by deleting gltS, pckA, and ptA genes to redirect carbon flux towards the TCA cycle, researchers achieved a 72-fold increase in glutamate concentration compared to wild type . In such engineered strains, ATP synthase function may be particularly important for maintaining energy homeostasis as the normal carbon flow is altered.

How does the ATP synthase of Shewanella contribute to its unique respiratory capabilities?

Shewanella species are distinguished by their remarkable respiratory versatility, capable of utilizing an exceptionally wide range of electron acceptors including oxygen, fumarate, and metal oxides like Fe(III) . ATP synthase plays a critical role in this respiratory flexibility through:

• Capturing energy from various electron transport chains with different thermodynamic properties

• Maintaining proton motive force under fluctuating environmental conditions

• Adapting to changes in proton gradient magnitude generated by different respiratory pathways

The ATP synthase must function across diverse bioenergetic regimes. For instance, under pyruvate fermentation conditions, S. oneidensis can survive without growth , suggesting ATP synthase may operate even at minimal proton gradients to provide maintenance energy. Conversely, during aerobic respiration, the ATP synthase must efficiently process the larger proton gradient generated by the oxygen-dependent electron transport chain.

What expression systems and purification strategies are optimal for recombinant Shewanella ATP synthase subunit c?

Recombinant Shewanella oneidensis ATP synthase subunit c (atpE) has been successfully expressed in E. coli expression systems, with the full-length protein (amino acids 1-83) fused to an N-terminal His tag . This approach facilitates purification while maintaining the protein's structural integrity.

For functional studies, it's critical to maintain the protein in a suitable detergent environment throughout purification to prevent aggregation of this highly hydrophobic protein. The final lyophilized product can be reconstituted in appropriate buffers for functional or structural studies .

How can researchers assess the assembly and functionality of recombinant ATP synthase components?

When working with individual subunits of the ATP synthase complex like atpE, assessing both assembly and functionality requires specialized approaches:

Assembly Assessment Methods:

• Size exclusion chromatography: To determine whether atpE oligomerizes correctly to form c-rings

• Native PAGE: To visualize intact c-rings and their association with other ATP synthase components

• Circular dichroism spectroscopy: To confirm the α-helical structure of reconstituted atpE

• Crosslinking studies: To capture transient interactions with neighboring subunits

Functionality Assessment Methods:

• Reconstitution into liposomes: To measure proton translocation activity

• ATP synthesis/hydrolysis assays: Using reconstituted ATP synthase complexes

• Proton flux measurements: Using pH-sensitive fluorescent dyes

For Shewanella ATP synthase specifically, researchers should consider the protein's natural operating conditions, including the pH range and ion concentrations that reflect the organism's native environment.

What techniques can be applied to study ATP synthase in the context of Shewanella's electron transfer pathways?

Shewanella's distinctive ability to transfer electrons to extracellular acceptors makes the study of ATP synthase in this context particularly interesting. Several techniques can be employed:

• Bioelectrochemical systems (BES): These systems can be used to quantify the relationship between electron transfer to external acceptors and ATP synthesis rates. By controlling the electrode potential, researchers can manipulate the energy available to the electron transport chain and measure the resulting ATP production through ATP synthase.

• Real-time monitoring of membrane potential: Using voltage-sensitive dyes or electrophysiology techniques to correlate electron transfer events with proton motive force generation that drives ATP synthase.

• Mutant analysis: Comparing ATP production in wild-type strains versus those with mutations in specific components of electron transfer pathways or ATP synthase genes. For example, creating strains with altered c-subunit stoichiometry to investigate how this affects the ATP/electron ratio.

• Genetic code expansion: As demonstrated with MtrC cytochrome, incorporating non-canonical amino acids into ATP synthase subunit c could enable site-specific labeling for visualizing its interactions with electron transport chain components .

How can ATP synthase engineering contribute to enhanced bioenergy applications of Shewanella?

Shewanella's unique respiratory capabilities make it valuable for bioenergy applications, particularly in microbial fuel cells and bioelectrochemical systems. Engineering the ATP synthase could enhance these applications through:

• Optimizing the c-ring stoichiometry: Altering the number of c-subunits in the ring changes the H⁺/ATP ratio, potentially allowing more efficient energy harvesting at lower proton motive force.

• Increasing proton conductance: Targeted mutations in the c-subunit could enhance proton flow, potentially increasing current production in bioelectrochemical systems.

• Engineering ATP synthase regulation: Modifying regulatory elements to maintain ATP synthase activity under fluctuating electrode potentials could improve the robustness of bioelectrochemical systems.

In metabolically engineered Shewanella strains, where carbon flux has been redirected towards valuable products like glutamate or itaconic acid , ATP synthase modifications could help balance energy demand with production efficiency.

What is the relationship between ATP synthase activity and pyruvate metabolism in Shewanella?

Shewanella displays interesting pyruvate metabolism patterns that interact with ATP synthase function. Under anaerobic conditions with limited electron acceptors, S. oneidensis MR-1 can ferment pyruvate for survival, though this does not support growth . The metabolic data reveals:

In the wild-type strain fermenting pyruvate, researchers observed production rates of 988.8 μmol/g AFDW·h for pyruvate, 926.6 μmol/g AFDW·h for acetate, 173.5 μmol/g AFDW·h for lactate, 556.3 μmol/g AFDW·h for formate, and 64.5 μmol/g AFDW·h for H₂ . These metabolic patterns suggest that ATP synthase activity is tightly coordinated with pyruvate metabolism pathways, with the relative contribution of oxidative phosphorylation varying based on electron acceptor availability.

What are promising approaches for integrating ATP synthase studies with genetic code expansion in Shewanella?

Combining ATP synthase research with genetic code expansion offers exciting possibilities for understanding energy metabolism in Shewanella. Future research could focus on:

• Site-specifically incorporating non-canonical amino acids at the c-ring/a-subunit interface to investigate proton translocation mechanisms.

• Using click chemistry-compatible amino acids in ATP synthase components to visualize assembly and distribution under different respiratory conditions.

• Incorporating photo-crosslinking amino acids to capture transient interactions between ATP synthase and other components of Shewanella's respiratory chains.

The demonstrated capability to incorporate diverse non-canonical amino acids like N^ε-Boc-l-lysine (BocK), N^ε-(4-pentynyloxycabonyl)-l-lysine (AlkK), and p-azido-l-phenylalanine (AzF) into Shewanella proteins provides a strong foundation for these approaches.

How might cross-disciplinary approaches advance our understanding of ATP synthase in Shewanella species?

Advancing our understanding of ATP synthase in Shewanella will likely require integration of:

• Structural biology: Cryo-EM and X-ray crystallography to determine Shewanella-specific features of ATP synthase structure.

• Synthetic biology: Applying genetic code expansion and metabolic engineering to create designer ATP synthase variants.

• Bioelectrochemistry: Understanding how electron flow to diverse acceptors couples with ATP synthase function.

• Systems biology: Integrating ATP synthase function into genome-scale metabolic models of Shewanella.

• Bioinformatics: Comparative analysis of ATP synthase components across Shewanella species adapted to different ecological niches.

Such interdisciplinary approaches will be particularly valuable for understanding how ATP synthase contributes to Shewanella's remarkable metabolic versatility and environmental adaptability.