Recombinant Escherichia coli O157:H7 ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c (atpE) in E. coli O157:H7?

ATP synthase subunit c forms the c-ring within the F₀ domain of ATP synthase, creating a critical component of the proton channel. This subunit is essential for the rotational mechanism that couples proton translocation across the membrane to ATP synthesis. In E. coli, the F₁F₀-ATPase catalyzes ATP synthesis from ADP and inorganic phosphate using the electrochemical gradient of protons across the cellular membrane . The greater pH gradient between intracellular and extracellular compartments, particularly in acidic environments (pH 5.5), can boost membrane potential and drive ATP synthesis . This functionality is especially relevant when studying pathogenic strains like O157:H7, which must adapt to acidic environments during host infection.

How does the structure of atpE contribute to ATP synthase function?

AtpE subunits assemble into a ring structure (c-ring) within the membrane domain of ATP synthase. Each c subunit contains two transmembrane helices and a conserved carboxylate residue (typically aspartate) that is essential for proton binding and translocation. The c-ring rotates during catalysis, driven by the proton motive force. This rotation is mechanically coupled to the central stalk of F₁, inducing conformational changes in the catalytic β subunits that lead to ATP synthesis . The bacterial F₁-ATPase structure reveals that the c-ring interacts with the ε subunit, which can adopt different conformations related to enzyme regulation and inhibition .

How is atpE expression regulated in response to environmental stress?

E. coli regulates ATP synthase expression in response to environmental conditions, particularly pH and energy availability. Under mildly acidic conditions (pH 5.5), E. coli increases its ATP concentration through multiple mechanisms. While one might expect upregulation of ATP synthase, research shows that genes related to oxidative phosphorylation are actually downregulated during acid stress, despite being major ATP producers at neutral pH . Instead, glycolytic enzymes (Glk, PykF, and Pgk) become the primary ATP producers under mild acidic stress . This metabolic shift allows the bacterium to maintain energy homeostasis while adapting to environmental challenges.

What is the optimal expression level of ATP synthase for E. coli growth?

Studies indicate that wild-type E. coli expresses H⁺-ATPase remarkably close (within a few percent) to optimal concentrations that maximize immediate growth rate across diverse conditions . This suggests that expression levels are finely tuned through evolution to optimize fitness. The expression pattern changes based on available carbon sources - during growth on sugars, when metabolism overflows with acetate, glycolysis supplies most ATP, while H⁺-ATPase becomes the main source of ATP synthesis during growth on acetate . This optimal expression profile likely extends to pathogenic strains like O157:H7, but may show strain-specific adaptations related to virulence and survival in host environments.

What are the most effective methods for recombinant expression of E. coli O157:H7 atpE?

For recombinant expression of E. coli O157:H7 atpE, researchers typically employ the following methodology:

• Gene amplification: PCR-amplify the atpE gene from E. coli O157:H7 genomic DNA using specific primers with appropriate restriction sites .

• Cloning: Insert the amplified gene into an expression vector like pET21-b with a suitable promoter (typically T7) and affinity tag (His-tag is common) .

• Host selection: Transform the construct into an appropriate E. coli expression strain like Rosetta(DE3)pLysS to address potential codon bias issues .

• Expression conditions: Induce protein expression with IPTG (0.5-1 mM) when cultures reach mid-log phase (OD₆₀₀ ≈ 0.6-0.8).

• Purification: Use affinity chromatography (HisTrap) for His-tagged proteins .

For membrane proteins like atpE, additional considerations include:

• Using mild detergents during cell lysis and purification

• Optimizing induction temperature (often lower temperatures like 18-25°C improve folding)

• Considering membrane fraction isolation protocols

What approaches can be used to assay ATP synthase activity in recombinant systems?

Several methodologies are available for assaying ATP synthase activity:

• ATP synthesis assay: Measure ATP production in membrane vesicles or reconstituted systems using luciferase-based bioluminescence assays (e.g., CLSII) . This approach can detect ATP at nanomolar concentrations.

• ATP hydrolysis assay: Measure inorganic phosphate release using colorimetric methods (malachite green or molybdate assays).

• Proton pumping assay: Monitor pH changes using pH-sensitive fluorescent dyes.

• Membrane potential measurements: Use voltage-sensitive dyes to track changes in membrane potential during enzyme activity.

• Single-molecule studies: Observe rotational dynamics of individual ATP synthase molecules using techniques referenced in studies of F₁ from E. coli .

How does the ε subunit interaction with c-ring affect ATP synthase regulation in E. coli O157:H7?

The ε subunit plays a crucial regulatory role in bacterial ATP synthases through interaction with the c-ring and other components. In E. coli, the C-terminal domain (CTD) of the ε subunit can adopt different conformations that directly impact enzyme activity . In the inhibitory state (ε X state), the extended conformation of the εCTD contacts five other subunits, with its terminal half inserted into the central cavity of F₁ . This arrangement correlates with an inactive enzyme state.

The interaction between ε subunit and c-ring (composed of atpE) is dynamic and depends on nucleotide conditions. The ε-inhibited state forms after nucleotide hydrolysis and appears to initiate at the catalytic dwell angle, with reversible rotation over approximately 40° involved in nucleotide effects on the inhibitory state of ε . This regulatory mechanism does not occur in mitochondrial ATP synthase, making it a potential target for antimicrobial development specifically against bacterial pathogens .

For researchers studying E. coli O157:H7, understanding these interactions could reveal strain-specific regulatory mechanisms that might contribute to pathogenicity or survival in host environments.

What are the implications of acid resistance mechanisms on ATP synthase function in E. coli O157:H7?

E. coli O157:H7's ability to survive gastric acidity is crucial for its pathogenicity. The relationship between acid resistance and ATP synthase function presents important research implications:

These findings suggest that O157:H7's enhanced acid resistance might involve specialized regulation of ATP synthase, potentially through strain-specific adaptations in the atpE subunit or its interactions with regulatory elements like the ε subunit.

What strategies can be employed to identify potential inhibitors of ATP synthase subunit c?

The development of inhibitors targeting bacterial ATP synthase subunit c represents an important research direction, as this subunit is distinct from its mitochondrial counterpart. A systematic approach includes:

• Structure-based virtual screening: Using homology modeling and molecular dynamics (MD) simulation to create refined models of atpE . This approach has been successful with Mycobacterium tuberculosis AtpE and could be applied to E. coli O157:H7.

• Database screening: Search chemical databases like Zinc and PubChem for compounds capable of binding to atpE with minimum binding energies using computational tools like RASPD and PyRx .

• Compound filtering: Screen potential inhibitors for favorable physicochemical properties using Lipinski's rule of five and perform molecular docking analysis to identify compounds with binding energies lower than ATP itself .

• ADME and toxicity analysis: Further screen promising compounds for absorption, distribution, metabolism, excretion, and toxicity properties .

• Experimental validation: Test candidate inhibitors using the ATP synthesis/hydrolysis assays described earlier.

How can recombinant systems be utilized for ATP regeneration in experimental setups?

Recombinant E. coli systems can be engineered to regenerate ATP for experimental applications using the following approach:

• Thermostable enzyme expression: Create recombinant E. coli expressing thermostable enzymes like Thermus polyphosphate kinase (PPK), which can regenerate ATP using inexpensive polyphosphate .

• Heat treatment protocol: Heat-treat the recombinant cells at 70°C for 10 minutes to inactivate host enzymes while retaining thermostable enzyme activity . This treatment also increases membrane permeability, allowing substrates and products to cross the cell membrane.

• Reaction setup: Add exogenous polyphosphate and ADP to the heat-treated cells to enable ATP regeneration . This system has shown >60% activity retention even after 1-week incubation at 70°C .

• Coupled enzyme reactions: Combine the ATP regeneration system with other thermostable enzymes for specific applications. For example, coupling with thermostable fructokinase and phosphofructokinase allows production of fructose 1,6-diphosphate from fructose and polyphosphate .

This approach overcomes inhibition issues associated with high ATP concentrations and provides a cost-effective ATP source, as commercial polyphosphate ($9/lb) can provide ATP equivalents that would cost over $2,000/lb if purchased directly .

How does E. coli O157:H7 ATP synthase subunit c differ from other bacterial species?

While specific sequence comparisons for E. coli O157:H7 atpE are not provided in the search results, general principles of bacterial ATP synthase conservation and divergence can be inferred:

What are the challenges in expressing functional recombinant ATP synthase in heterologous systems?

Researchers face several challenges when expressing ATP synthase components in heterologous systems:

• Multi-subunit assembly: ATP synthase is a complex multi-subunit enzyme. Expressing just the c subunit (atpE) may not provide insights into its function within the assembled complex.

• Membrane protein expression: As a membrane protein, atpE presents challenges typical of membrane protein expression, including potential toxicity to host cells, inclusion body formation, and difficulties in proper membrane insertion.

• Post-translational modifications: Any required post-translational modifications must be correctly performed by the host system.

• Functional reconstitution: For functional studies, the expressed protein must be properly reconstituted into a membrane environment that allows proton translocation and rotation.

• Species compatibility: When expressing components from different species together (as in chimeric enzymes), structural incompatibilities may prevent proper assembly or function.

Researchers have addressed some of these challenges using approaches like heat-treated recombinant systems , which allow for simplified experimental setups while retaining specific enzymatic functions.