Recombinant Acidovorax citrulli ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in Acidovorax citrulli?

ATP synthase subunit c (atpE) is a critical component of the F₀F₁-ATP synthase complex in A. citrulli. It forms part of the membrane-embedded F₀ portion, specifically the c-ring that facilitates proton translocation across the membrane. This proton movement drives the rotation of the c-ring, which in turn drives the synthesis of ATP in the F₁ portion of the complex. The c-subunit contains an essential proton-binding carboxyl group that functions as the proton carrier, allowing the protein to bind and transport protons across the membrane . This energy conversion mechanism is fundamental to bacterial bioenergetics and survival.

How does the structure of A. citrulli ATP synthase subunit c compare to that of other bacterial species?

While specific structural data on A. citrulli ATP synthase subunit c is limited, the c-subunit structure is relatively conserved across bacterial species with some notable variations. The c-ring is composed of 8-17 c-subunits depending on the species . Each c-subunit typically contains two transmembrane α-helical domains connected by a polar loop, with the essential proton-binding carboxyl group (usually glutamate or aspartate) located near the center of the membrane-embedded region. This structural arrangement enables the protein to participate in the proton translocation mechanism necessary for ATP synthesis.

What is known about the stoichiometry of ATP synthase c-ring in Acidovorax citrulli?

While the exact stoichiometry of the c-ring in A. citrulli has not been definitively established in the provided research, c-ring stoichiometry generally varies from 8 to 15 subunits among different bacterial species . This variation has direct implications for the bioenergetics of the organism, as each c-subunit binds and transports one H⁺ across the membrane during a complete rotation. The c-ring rotation drives the rotation of the gamma-subunit, which results in the synthesis of 3 ATP molecules per complete rotation . Therefore, the c-ring stoichiometry determines the H⁺/ATP ratio, a crucial parameter for understanding the energy efficiency of the bacterium.

What are the optimal expression systems for recombinant A. citrulli ATP synthase subunit c?

Based on similar research with other bacterial ATP synthase c-subunits, E. coli expression systems have proven effective for recombinant production. For optimal expression, researchers should consider:

• Codon optimization: The gene sequence should be optimized for expression in E. coli to overcome potential codon bias issues .

• Fusion protein approach: Expressing the c-subunit as a fusion with a larger, more soluble protein such as maltose binding protein (MBP) can significantly improve expression yields and solubility .

• Expression conditions: Optimization of induction temperature (typically 18-30°C), inducer concentration, and duration is critical to balance protein expression with proper folding.

• Vector selection: pET-based vectors with strong T7 promoters or similar expression systems often provide good yields of membrane proteins.

This approach has been successfully used for expressing c-subunits from other species and should be adaptable to A. citrulli atpE expression .

What purification strategies are most effective for recombinant A. citrulli ATP synthase subunit c?

Purification of the highly hydrophobic ATP synthase subunit c requires specialized techniques. An effective purification strategy based on successful approaches with other c-subunits includes:

• Affinity chromatography: If expressed as a fusion protein (e.g., MBP-atpE), initial purification can be performed using affinity chromatography specific to the fusion partner .

• Protease cleavage: The fusion protein can be cleaved by a specific protease in the presence of a detergent to release the c-subunit while maintaining its solubility .

• Reversed-phase chromatography: Final purification can be achieved using reversed-phase column chromatography with ethanol as an eluent, which has proven effective for similar c-subunits .

• Quality control: Circular dichroism spectroscopy should be used to confirm that the purified c-subunit maintains its native alpha-helical secondary structure .

This multi-step approach helps overcome the challenges associated with purifying highly hydrophobic membrane proteins while maintaining their structural integrity.

How can researchers verify the proper folding and oligomerization of recombinant A. citrulli ATP synthase subunit c?

Verifying proper folding and oligomerization is crucial for functional studies. Researchers should employ multiple complementary techniques:

• Circular dichroism (CD) spectroscopy: This provides clear evidence of proper alpha-helical secondary structure, which is characteristic of correctly folded c-subunits .

• Size exclusion chromatography: This can help determine whether the c-subunits form oligomers of the expected size.

• Native PAGE: This can provide information about the oligomeric state under non-denaturing conditions.

• Reconstitution in liposomes: Functional c-rings can be reconstituted in liposomes, and their oligomeric state can be assessed through functional assays or imaging techniques .

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry can provide insights into the structural arrangement of c-subunits within the oligomeric ring.

These methods collectively provide robust verification of proper folding and oligomerization, which are prerequisites for functional studies.

What methods are available for characterizing the proton translocation function of recombinant A. citrulli ATP synthase subunit c?

Characterizing the proton translocation function requires reconstitution of the c-subunit into membrane systems. Key methodological approaches include:

• Liposome reconstitution: Purified c-subunits can be reconstituted into liposomes containing pH-sensitive fluorescent dyes to monitor proton translocation .

• Minimal respiratory chain reconstitution: A functional assay can be designed by co-reconstituting the c-ring with other components of ATP synthase and a pmf-generating terminal oxidase in liposomes .

• Patch-clamp techniques: These can be used to directly measure proton conductance across membranes containing reconstituted c-rings.

• pH jump experiments: These can assess the ability of the c-ring to respond to pH gradients.

• ATP synthesis assays: When reconstituted with the complete F₁F₀-ATP synthase, ATP synthesis rates can be measured in response to artificially imposed proton gradients.

These approaches provide complementary information about the proton translocation function and can be adapted to study specific aspects of the c-subunit's role in energy conversion.

How can researchers study the interactions between A. citrulli ATP synthase subunit c and other subunits of the F₀F₁ complex?

Studying protein-protein interactions within the ATP synthase complex requires specialized techniques suitable for membrane proteins:

• Co-immunoprecipitation: Using antibodies against one subunit to pull down interacting partners.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry can identify proximity relationships between subunits.

• Yeast two-hybrid membrane systems: Modified yeast two-hybrid systems designed for membrane proteins can detect direct interactions.

• Förster resonance energy transfer (FRET): This can assess proximity and interactions between fluorescently labeled subunits.

• Cryo-electron microscopy: This provides structural information about the assembled complex, revealing the interfaces between subunits.

• Molecular dynamics simulations: These can provide insights into the dynamic interactions between subunits during the rotation cycle .

These methods provide complementary information about the interaction network within the ATP synthase complex, helping to understand how the c-subunit functions in the context of the complete enzyme.

How can studies of A. citrulli ATP synthase subunit c contribute to understanding bacterial pathogenesis?

Research on A. citrulli ATP synthase subunit c can provide valuable insights into bacterial pathogenesis through several avenues:

• Energy metabolism during infection: Understanding how A. citrulli maintains energy production during different phases of infection can reveal adaptation strategies for survival in the host environment.

• Stress response mechanisms: ATP synthase function may be modulated during stress conditions encountered during infection, providing insights into bacterial adaptation to host defenses.

• Potential drug targets: As an essential component of energy metabolism, the ATP synthase represents a potential target for novel antimicrobials specific to A. citrulli.

• Evolutionary adaptations: Comparative studies of ATP synthase subunits across different bacterial pathogens can reveal evolutionary adaptations to specific host environments.

• Metabolic integration: Understanding how ATP production is integrated with virulence factor expression can reveal regulatory networks important for pathogenesis.

These research directions can complement studies on more traditional virulence factors like type III secretion effectors (e.g., AopU, AopV) that directly interact with host immunity .

What are the implications of c-ring stoichiometry variation for A. citrulli bioenergetics?

The stoichiometry of the c-ring has profound implications for bacterial bioenergetics and adaptation:

• H⁺/ATP ratio: The number of c-subunits in the ring directly determines the number of protons required to synthesize three ATP molecules, affecting the energetic efficiency of the bacterium .

• Adaptation to different environments: Variations in c-ring stoichiometry may represent adaptations to different environmental conditions, particularly to the magnitude of the proton motive force available in different habitats.

• Growth rate implications: The energetic efficiency determined by c-ring stoichiometry can influence growth rates and competitive fitness in different environments.

• Metabolic flexibility: The relationship between c-ring stoichiometry and ATP yield may influence the bacterium's ability to utilize different metabolic pathways.

Understanding these relationships can provide insights into A. citrulli's metabolic adaptation to its ecological niche .

How does the cooperation among c-subunits contribute to rotation mechanics in A. citrulli ATP synthase?

Advanced research on c-subunit cooperation reveals sophisticated mechanical principles:

• Sequential protonation/deprotonation: Research suggests that multiple c-subunits at the a/c interface cooperate during c-ring rotation .

• Deprotonated state distribution: Studies indicate that two or three deprotonated carboxyl residues may face the a-subunit simultaneously, with the waiting time for proton uptake shared between these subunits .

• Positional effects: The relative position of functional c-subunits appears to influence activity, with closer subunits showing higher cooperative function .

• Structural constraints: The spatial arrangement of cooperating c-subunits is likely constrained by the structure of the a/c interface.

• Energy landscape: The cooperation among c-subunits creates a complex energy landscape that guides the rotation in the proper direction.

These principles of cooperative function likely apply to A. citrulli ATP synthase and represent an important area for future research to understand the molecular mechanics of this sophisticated molecular machine .

What methodological approaches can be used to study the effects of mutations in A. citrulli ATP synthase subunit c?

Studying the effects of mutations in the c-subunit requires a comprehensive set of techniques:

• Site-directed mutagenesis: Creating specific mutations, particularly in the essential proton-binding carboxyl group (e.g., glutamate to glutamine mutations), to assess their impact on function .

• Bacterial complementation: Introducing mutated versions of atpE into knockout strains to assess functional complementation.

• In vitro reconstitution: Reconstituting mutant c-subunits into liposomes to assess their ability to form functional c-rings and conduct protons .

• ATP synthesis/hydrolysis assays: Measuring the impact of mutations on ATP synthesis or hydrolysis rates.

• Molecular dynamics simulations: Computational approaches to predict how mutations affect proton binding, protein dynamics, and subunit interactions .

• Structural studies: Comparing the structures of wild-type and mutant c-rings to understand the molecular basis of functional changes.

These approaches can reveal the structure-function relationships within the c-subunit and identify critical residues for ATP synthase function.

How might understanding A. citrulli ATP synthase contribute to synthetic biology applications?

ATP synthase research has significant implications for synthetic biology:

• Minimal energy systems: Understanding the minimal requirements for ATP production can inform the design of synthetic energy systems for artificial cells .

• Altered stoichiometry: Engineering c-rings with altered stoichiometry could create synthetic systems with customized H⁺/ATP ratios for specific applications.

• Alternative energy sources: Integration of ATP synthase with alternative energy-capturing mechanisms, similar to the hydrogenase-driven system described in some bacteria .

• Biosensors: ATP synthase components could be adapted as biosensors for proton gradients or energy status.

• Nanomotors: The rotary mechanism of ATP synthase could inspire the design of synthetic nanomotors for various applications.

These applications represent the translation of fundamental research on ATP synthase into practical synthetic biology tools and systems.

What are the major technical challenges in studying recombinant A. citrulli ATP synthase subunit c?

Researchers face several significant challenges when studying this membrane protein:

• Expression and purification difficulties: The highly hydrophobic nature of the c-subunit makes it challenging to express in sufficient quantities and purify in a properly folded state .

• Functional reconstitution: Reconstituting the c-subunit into functional c-rings that properly integrate into membrane systems requires optimization of lipid composition and reconstitution conditions .

• Stoichiometry determination: Accurately determining the native stoichiometry of the A. citrulli c-ring is technically challenging and may require specialized structural biology techniques.

• Functional assays: Developing sensitive assays to measure the proton translocation function of the isolated c-ring presents methodological challenges.

• Coordination with other subunits: Understanding how the c-ring coordinates with other ATP synthase subunits during rotation requires complex experimental setups.

Addressing these challenges requires innovative methodological approaches and integration of multiple complementary techniques.

How might A. citrulli ATP synthase research contribute to understanding bacterial adaptation to environmental stress?

ATP synthase function is likely central to stress adaptation:

• pH stress adaptation: The c-subunit's proton-binding properties may be optimized for the pH ranges encountered during different stages of infection.

• Energy conservation during nutrient limitation: ATP synthase efficiency may be crucial during periods of nutrient starvation, similar to the "lifeline" energy conservation mechanisms described in other bacteria .

• Temperature adaptation: The thermal stability and function of the c-ring may reflect adaptation to the temperature ranges encountered during the A. citrulli lifecycle.

• Host defense response: Maintaining energy production during exposure to host defense mechanisms may involve regulated changes in ATP synthase activity.

• Biofilm formation: Energy requirements during different stages of biofilm formation may influence ATP synthase expression and function.

Research in these areas can reveal how this fundamental enzyme has been adapted to support the specific lifestyle and pathogenicity of A. citrulli.

What novel approaches might advance our understanding of the relationship between ATP synthase function and A. citrulli virulence?

Several innovative approaches could reveal connections between energy metabolism and virulence:

• Conditional mutants: Developing conditional atpE mutants to study the relationship between ATP production and virulence factor expression under different conditions.

• In vivo imaging: Using fluorescently tagged ATP synthase components to track their localization and activity during different stages of infection.

• Metabolic flux analysis: Integrating ATP synthase function with global metabolic networks to understand how energy production supports virulence.

• Comparative genomics: Examining variations in ATP synthase components across different A. citrulli strains with varying virulence.

• Host-pathogen interface studies: Investigating whether ATP synthase function is modulated in response to host defense mechanisms, similar to how type III effectors like AopU and AopV interact with host immunity .

• Systems biology approaches: Integrating transcriptomic, proteomic, and metabolomic data to understand the regulatory networks connecting energy metabolism and virulence.

These approaches can provide a more integrated understanding of how fundamental cellular processes like energy production contribute to the pathogenic potential of A. citrulli.