Recombinant Aeromonas hydrophila subsp. hydrophila ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c (atpE) in Aeromonas hydrophila?

ATP synthase subunit c (atpE) in A. hydrophila is a small hydrophobic protein component of the F0 sector of ATP synthase, functioning as part of the membrane-embedded proton channel. According to sequence data, it consists of 80 amino acids with the sequence: MENLNMDLLYIAAAMMMGLAAIGASIGIGILGGKFLEGAARQPDLIPVLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVAG . This highly hydrophobic protein forms an oligomeric ring structure in the bacterial membrane that rotates during ATP synthesis, driven by the proton gradient. The protein's hydrophobic nature is evidenced by its high content of glycine, leucine, isoleucine, and alanine residues, which facilitate its integration into the lipid bilayer.

How does A. hydrophila atpE compare with ATP synthase subunit c from other bacterial species?

When comparing A. hydrophila atpE with other bacterial homologs, researchers should conduct sequence alignment analysis using tools like BLAST or Clustal Omega. A. hydrophila is a gram-negative bacterium that shares evolutionary history with other proteobacteria, particularly vibrios, as revealed in genome studies . The ATP synthase complex is highly conserved across bacterial species, but key amino acid differences in the c-subunit can affect proton binding, oligomerization, and inhibitor sensitivity. These variations may correlate with the organism's adaptation to different environmental conditions, as A. hydrophila has evolved to thrive in various aquatic environments, including polluted waters .

What genomic context surrounds the atpE gene in A. hydrophila?

The atpE gene in A. hydrophila ATCC 7966 strain is designated by the locus tag AHA_4267 . Based on typical bacterial ATP synthase organization, the atpE gene likely exists as part of the atp operon, which encodes all components of the F1F0 ATP synthase. In most bacteria, the atp genes are arranged in a conserved order. The A. hydrophila genome is approximately 4.7-Mb in size and contains numerous genes contributing to its versatile metabolism and virulence potential . Researchers should analyze the genomic context to understand potential co-regulation with other energy metabolism genes, which could provide insights into the bacterium's bioenergetic adaptations.

What are the optimal conditions for recombinant expression of A. hydrophila atpE?

For recombinant expression of A. hydrophila atpE, researchers should consider the following methodological approach:

• Expression system selection: Due to the hydrophobic nature of atpE, membrane protein expression systems are recommended. E. coli strains C41(DE3) or C43(DE3), specifically designed for membrane protein expression, are suitable hosts.

• Vector design: Incorporate a removable tag (His-tag or GST) to facilitate purification. Similar approaches have been successful with other A. hydrophila proteins, such as GalE, which yielded high expression levels in E. coli .

• Expression conditions: Based on patterns established with other A. hydrophila proteins, induction with 0.5-1.0 mM IPTG at mid-log phase (OD600 of 0.6-0.8) followed by overnight expression at 18-20°C typically minimizes inclusion body formation.

• Codon optimization: Consider codon optimization for E. coli, especially for rare codons, to enhance expression levels.

Similar membrane proteins from A. hydrophila have been successfully expressed as recombinant proteins, indicating that established protocols can be adapted for atpE .

What purification strategies are most effective for isolating recombinant A. hydrophila atpE?

Purification of recombinant atpE requires specialized techniques due to its hydrophobic nature:

• Membrane fraction isolation: After cell disruption, collect the membrane fraction by ultracentrifugation (typically 100,000×g for 1 hour).

• Detergent solubilization: Screen multiple detergents for optimal solubilization. Common options include n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucopyranoside (OG), or lauryl maltose neopentyl glycol (LMNG) at concentrations of 1-2%.

• Affinity chromatography: If using His-tagged constructs, Ni²⁺-NTA affinity chromatography is effective, as demonstrated with other A. hydrophila recombinant proteins where yields of ~112 mg/L have been achieved .

• Size exclusion chromatography: For further purification and to verify the oligomeric state, size exclusion chromatography in the presence of detergent is recommended.

• Tag removal: If native protein is required, consider enzymatic cleavage of the tag followed by reverse affinity chromatography, similar to the approach used for tag-less GalE purification from A. hydrophila .

Expected yields may vary, but based on other membrane proteins, 5-15 mg/L of purified protein from shake flask cultures is a reasonable target.

How can the stability and solubility of recombinant A. hydrophila atpE be maximized?

To maximize stability and solubility of recombinant atpE:

• Buffer optimization: Screen various buffers (pH 6.5-8.0), salt concentrations (100-500 mM NaCl), and stabilizing agents (glycerol 5-20%).

• Detergent screening: Systematic testing of detergent types and concentrations is crucial. Create a detergent screening matrix:

• Lipid supplementation: Addition of E. coli polar lipid extract (0.01-0.05 mg/mL) or specific phospholipids can enhance stability.

• Storage conditions: For extended storage, maintain the protein at -20°C or -80°C in the presence of 50% glycerol . Avoid repeated freeze-thaw cycles.

• Stabilizing mutations: Consider introducing strategic mutations that enhance stability without affecting function, guided by comparative sequence analysis with more stable homologs.

What techniques are most appropriate for studying the structural properties of A. hydrophila atpE?

For structural characterization of atpE, researchers should consider these complementary techniques:

• Cryo-electron microscopy (cryo-EM): Most suited for visualizing the entire ATP synthase complex, including the c-ring formed by atpE subunits. Sample preparation would involve purification in amphipols or nanodiscs rather than detergent micelles.

• X-ray crystallography: Challenging but possible for the c-ring structure. Crystallization trials should explore lipidic cubic phase (LCP) methods which have proven successful for other membrane proteins.

• Solid-state NMR: Particularly valuable for analyzing the dynamics of atpE within lipid bilayers. Sample preparation would involve reconstitution in isotopically labeled lipid bilayers.

• Circular dichroism (CD) spectroscopy: Useful for confirming secondary structure content, which is expected to show high alpha-helical content. Typical protocol involves scanning 190-260 nm in detergent solution.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Provides information on solvent accessibility and conformational dynamics. This would be particularly valuable for identifying regions involved in proton transport.

A combined approach using multiple techniques would provide the most comprehensive structural understanding of this membrane protein.

How can the functional properties of A. hydrophila atpE be assessed in vitro?

Functional characterization of atpE requires specialized biophysical and biochemical approaches:

• Proton transport assays: Reconstitute purified atpE into liposomes loaded with pH-sensitive fluorophores (e.g., ACMA or pyranine). Monitor fluorescence changes upon establishment of a proton gradient.

• ATP synthesis activity: Measure ATP production by reconstituted ATP synthase complexes containing atpE using luciferase-based ATP detection assays.

• Inhibitor binding studies: Assess binding of known ATP synthase inhibitors (e.g., oligomycin, DCCD) using isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR).

• Site-directed mutagenesis: Introduce mutations at key conserved residues (particularly the essential proton-binding glutamate/aspartate) and assess functional consequences. This approach has been used successfully with other A. hydrophila recombinant proteins to understand structure-function relationships .

• Oligomerization analysis: Use analytical ultracentrifugation or native PAGE to determine the number of atpE subunits in the c-ring, which can vary between bacterial species and influences the bioenergetic properties of ATP synthase.

These methodologies can reveal unique functional adaptations of A. hydrophila atpE compared to homologs from other bacteria.

What computational approaches can provide insights into A. hydrophila atpE function?

Computational methods offer valuable insights into atpE structure and function:

• Homology modeling: Generate 3D structural models based on known crystal structures of c-subunits from other bacteria. Tools like SWISS-MODEL or Phyre2 are suitable, followed by energy minimization in a simulated membrane environment.

• Molecular dynamics simulations: Perform simulations of atpE within a lipid bilayer to understand conformational dynamics and proton transport mechanisms. For A. hydrophila atpE, which functions in variable environments, simulate conditions of different pH and ionic strength.

• Coevolution analysis: Tools like EVcouplings can identify residues that have co-evolved, providing insights into structural contacts and functional relationships within the protein.

• Electrostatic surface mapping: Calculate the electrostatic potential around the protein to identify potential proton pathways and binding sites.

• Prediction of post-translational modifications: Analyze the sequence for potential modification sites that might regulate function.

These computational approaches should be iteratively combined with experimental validation to develop a comprehensive understanding of atpE function.

How does atpE contribute to the pathogenicity and survival of A. hydrophila in different environments?

ATP synthase subunit c plays a crucial role in A. hydrophila's adaptability and pathogenicity:

• Energy metabolism: As a component of ATP synthase, atpE is essential for energy production, enabling A. hydrophila to thrive in diverse environments. The genome of A. hydrophila reveals a physiologically adroit organism with broad metabolic capabilities , suggesting that ATP synthesis is optimized for different conditions.

• pH tolerance: The proton-binding properties of atpE may contribute to A. hydrophila's ability to survive in environments with varying pH, which is relevant to its pathogenicity in both aquatic hosts and human tissues.

• Antimicrobial resistance: ATP synthase can be a target for certain antimicrobials. Studies on other A. hydrophila proteins have noted increasing antibiotic resistance issues , suggesting that understanding energy metabolism components like atpE could reveal resistance mechanisms.

• Virulence factor expression: Energy production is linked to the expression of virulence factors. A. hydrophila possesses a large array of virulence genes , and ATP synthesis likely supports the energy requirements for their expression.

• Biofilm formation: ATP synthesis supports the energy-intensive process of biofilm formation, which is important for A. hydrophila survival in aquatic environments and medical devices.

Research on recombinant atpE can provide insights into these aspects of A. hydrophila biology.

Could A. hydrophila atpE serve as a target for antimicrobial development?

ATP synthase is an emerging target for antimicrobial development, and A. hydrophila atpE shows potential in this regard:

• Essential function: As a component of the ATP synthesis machinery, atpE is essential for bacterial survival, making it a promising drug target.

• Structural uniqueness: Any structural differences between bacterial and host ATP synthase could be exploited for selective targeting. The 80-amino acid sequence of A. hydrophila atpE should be analyzed for unique features compared to eukaryotic homologs.

• Established inhibitors: Known ATP synthase inhibitors like oligomycin and DCCD could serve as starting points for developing A. hydrophila-specific compounds.

• Alternative to conventional antibiotics: Given the problems of antibiotic resistance reported in A. hydrophila , ATP synthase inhibitors could offer an alternative therapeutic approach.

• Combination therapy potential: ATP synthase inhibitors could potentially synergize with existing antibiotics or phage therapy approaches, which have shown promise against A. hydrophila infections .

Research using recombinant atpE could facilitate screening of small molecule libraries for novel inhibitors with specificity for A. hydrophila ATP synthase.

What role might A. hydrophila atpE play in adaptation to environmental stressors?

A. hydrophila inhabits diverse aquatic environments, suggesting that atpE may have evolved specific adaptations:

• Temperature adaptation: A. hydrophila survives in various temperature conditions, suggesting that its ATP synthase, including atpE, may have evolved temperature-stability features. Thermal stability assays with recombinant atpE can reveal these adaptations.

• Response to oxygen levels: As a facultative anaerobe, A. hydrophila must adapt its energy metabolism to varying oxygen conditions. atpE may show structural or functional adaptations that optimize ATP synthesis under different redox conditions.

• Osmotic stress response: A. hydrophila has been found in environments ranging from freshwater to brackish water, suggesting that atpE functions across a range of osmotic conditions. Studies with other A. hydrophila proteins have shown their role in survival under different osmolaric conditions .

• pH tolerance mechanisms: The proton-binding properties of atpE may contribute to pH homeostasis in acidic or alkaline environments.

• Heavy metal resistance: A. hydrophila possesses resistance mechanisms against toxic compounds encountered in polluted waters , and energy-dependent efflux systems powered by ATP synthase may contribute to this resistance.

Comparative studies of recombinant atpE under different environmental conditions can elucidate these adaptive mechanisms.

How can CRISPR-Cas9 technology be applied to study the role of atpE in A. hydrophila physiology?

CRISPR-Cas9 technology offers powerful approaches for studying atpE function in vivo:

• Gene knockout challenges: Complete knockout of atpE would likely be lethal, necessitating conditional knockout strategies such as:

  • Inducible promoter control of atpE expression

  • CRISPRi (CRISPR interference) for partial suppression

  • Temperature-sensitive variants

• Point mutation strategy: Rather than complete knockout, introduce specific mutations in conserved residues to study functional consequences:

  • Mutations in the essential proton-binding site

  • Alterations in residues involved in c-ring assembly

  • Modifications at the interface with other ATP synthase subunits

• Reporter system integration: Fuse fluorescent reporters to atpE or its promoter to monitor expression patterns under different environmental conditions.

• Complementation studies: Create knockout strains complemented with atpE variants from other species to identify specific adaptations of A. hydrophila atpE.

• Whole-genome phenotypic screening: Perform CRISPR screening to identify genetic interactions with atpE, revealing metabolic networks dependent on ATP synthase function.

These approaches can reveal the physiological significance of atpE in the context of A. hydrophila's complex metabolism and virulence potential .

What are the best approaches for studying potential post-translational modifications of A. hydrophila atpE?

Post-translational modifications (PTMs) of atpE could regulate ATP synthase function in A. hydrophila:

• Mass spectrometry protocols:

  • Employ high-resolution LC-MS/MS after enrichment for modified peptides

  • Use multiple proteases (not just trypsin) to ensure coverage of hydrophobic regions

  • Apply electron transfer dissociation (ETD) fragmentation, which preserves labile modifications

• Modification-specific enrichment:

  • Phosphorylation: Use titanium dioxide or immobilized metal affinity chromatography

  • Acetylation: Apply anti-acetyllysine antibodies for immunoprecipitation

  • Oxidative modifications: Use appropriate redox proteomics approaches

• Site-directed mutagenesis validation:

  • Mutate identified modification sites to non-modifiable residues

  • Assess functional consequences of preventing modification

  • Create modification-mimicking mutations (e.g., phosphomimetic)

• Temporal dynamics:

  • Study modification patterns under different growth conditions

  • Monitor modification changes during infection processes

  • Examine modifications during environmental stress responses

• Enzymatic regulation:

  • Identify kinases/phosphatases that may target atpE

  • Study acetyltransferases/deacetylases that could modify atpE

  • Investigate regulatory proteins that interact with modified atpE

These approaches will reveal how A. hydrophila regulates ATP synthase activity in response to environmental changes.

What potential biotechnological applications could be developed from recombinant A. hydrophila atpE?

Recombinant atpE from A. hydrophila offers several biotechnological possibilities:

• Nanomotor development: ATP synthase c-rings function as molecular rotary motors and could be engineered for nanotechnological applications:

  • Immobilized on synthetic surfaces to create nanoscale mechanical devices

  • Integrated into biomimetic systems for energy conversion

  • Modified to respond to different ion gradients or stimuli

• Biosensor creation: The proton-binding properties of atpE could be exploited to develop sensors for:

  • Local pH changes in microenvironments

  • Membrane potential fluctuations

  • Proton gradient formation in artificial systems

• Biomimetic energy systems: Understanding the efficiency of A. hydrophila ATP synthase could inspire:

  • Improved fuel cell designs

  • Novel approaches to biological energy storage

  • Artificial photosynthetic systems with enhanced efficiency

• Drug screening platform: Recombinant atpE could serve as a target for screening:

  • Novel antibiotics specific to A. hydrophila

  • Compounds that inhibit bacterial bioenergetics

  • Molecules that selectively disrupt bacterial membrane proteins

• Protein engineering platform: The robust expression system developed for A. hydrophila atpE could be adapted for other challenging membrane proteins, particularly those with industrial or pharmaceutical significance.

These applications extend beyond the immediate research on A. hydrophila pathogenicity to broader biotechnological innovations.

What are the most significant challenges in working with recombinant A. hydrophila atpE?

Researchers face several key challenges when working with recombinant atpE:

• Membrane protein expression barriers: The hydrophobic nature of atpE presents challenges for high-yield expression, requiring optimization of expression systems, detergents, and purification protocols. Similar challenges have been reported with other A. hydrophila membrane proteins .

• Functional reconstitution complexity: Ensuring that recombinant atpE retains its native structure and function requires careful reconstitution into suitable membrane mimetics.

• Structural determination difficulties: Obtaining high-resolution structural data for membrane proteins like atpE requires specialized approaches and often faces technical hurdles.

• Physiological context limitations: In vitro studies may not fully recapitulate the complex physiological environment in which atpE functions in A. hydrophila.

• Translational barriers: Bridging fundamental research on atpE to applications such as drug development or vaccine design requires interdisciplinary approaches.

Despite these challenges, the systematic approaches outlined in this FAQ collection provide pathways to overcome these obstacles and advance our understanding of this important bacterial protein.

How might research on A. hydrophila atpE contribute to broader understanding of bacterial bioenergetics?

Research on A. hydrophila atpE has significant implications for bacterial bioenergetics:

• Comparative bioenergetics: A. hydrophila's ability to thrive in diverse environments suggests its ATP synthase may have unique adaptations. Comparative studies with other bacterial species could reveal evolutionary principles in bioenergetic systems.

• Environmental adaptation mechanisms: Understanding how atpE functions across environmental conditions can illuminate how bacteria adapt their energy metabolism to stress.

• Pathogen-specific bioenergetic features: Identifying unique aspects of A. hydrophila ATP synthase could reveal how energy metabolism supports pathogenicity, potentially applicable to other bacterial pathogens.

• Structure-function relationships: Detailed studies of atpE structure and function can enhance our fundamental understanding of proton translocation and rotary catalysis mechanisms.

• Energy conservation strategies: Analysis of A. hydrophila atpE efficiency could provide insights into bacterial energy conservation under resource-limited conditions.