Recombinant Escherichia coli O6:K15:H31 ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in bacterial energy metabolism?

ATP synthase is one of the key enzymes involved in both photosynthesis and cellular respiration. In E. coli, the subunit c forms a ring (cn) in the F0 region of ATP synthase. This rotation is coupled to the γ-stalk rotation in the F1 region, where subunit γ functions as a shaft inside the α3β3 head. The process drives the catalysis of ATP synthesis that occurs at the three α-β interfaces in F1. This coordinated mechanism produces 3 ATP molecules for every n protons that pass through the membrane, where n represents the number of c subunits in the ring . The entire process is reversible in F-type ATP synthases, allowing both ATP synthesis and hydrolysis depending on cellular energy requirements.

What is the significance of the c-ring stoichiometry in ATP synthase function?

The number of c subunits forming the ring (cn) varies among organisms, creating important functional differences in energy conversion efficiency. The table below illustrates this variation:

The coupling ratio (ions transported:ATP generated) is determined by n/3, as 3 ATP molecules are synthesized per complete c-ring rotation regardless of size . This stoichiometric variation directly impacts the bioenergetic efficiency of the organism, with potential evolutionary adaptations to different environmental niches and energy requirements . The biological significance of this variation remains an active area of research with several competing hypotheses .

How does the atpE intercistronic sequence enhance translational efficiency?

The 30-bp intercistronic sequence found immediately upstream of the E. coli atpE gene Shine-Dalgarno (SD) sequence dramatically improves translational efficiency . Studies demonstrate this sequence increases expression of both native and foreign genes by a factor of 6-10 when added to their translational initiation regions . The sequence comprises a U-rich region followed by an interrupted A-rich region (UUUUAACUGAAACAAA) that likely functions as a specific recognition signal for the E. coli translational apparatus .

This enhancement appears independent of mRNA stability or secondary structure effects. Importantly, the translational enhancement occurs even with genes having codon usage typically associated with weak expression in E. coli, suggesting the sequence's primary structure serves as a direct recognition element for translation machinery rather than affecting local mRNA conformation . This finding has significant implications for recombinant protein production strategies in E. coli expression systems.

What factors influence atpE gene expression levels in different E. coli strains?

Several factors impact atpE expression across E. coli strains:

• Ribosome binding site (RBS) sequence: The presence of the atpE-specific RBS pattern (U-rich sequence followed by interrupted A-rich sequence) significantly enhances translational efficiency .

• mRNA stability: The functional half-life of mRNA constructs containing the atpE RBS is approximately 2.8 minutes, compared to 2.4 minutes for constructs lacking this element .

• Promoter strength: Expression vectors utilizing strong, inducible promoters like PRPL show optimal expression of constructs containing atpE elements .

• Strain-specific factors: E. coli strains DH1 and MCG1 have been successfully used for expression studies involving atpE and its regulatory elements .

• Genetic context: The enhancement effect of the atpE sequence is transferable to other genes, indicating its function as a general translational enhancer rather than a gene-specific element .

These factors must be considered when designing expression systems for recombinant protein production utilizing atpE regulatory elements.

What are the optimal methods for isolating and purifying recombinant atpE from E. coli?

Purification of recombinant atpE presents unique challenges due to its hydrophobic nature and membrane localization. An effective purification protocol should include:

• Cell lysis optimization: Mechanical disruption (sonication or high-pressure homogenization) in buffers containing protease inhibitors to prevent degradation.

• Membrane fraction isolation: Differential centrifugation to separate membrane fractions containing the expressed atpE protein.

• Detergent solubilization: Carefully selected detergents (mild non-ionic such as DDM or LDAO) to solubilize atpE while maintaining native structure.

• Affinity chromatography: If using tagged constructs, immobilized metal affinity chromatography (IMAC) for histidine-tagged proteins or other appropriate affinity methods.

• Size exclusion chromatography: To achieve higher purity and separate different oligomeric states.

• Functional validation: ATP synthesis or hydrolysis assays to confirm biological activity of the purified protein.

Researchers should conduct small-scale optimization experiments before scaling up, as membrane protein purification conditions often require strain-specific adjustments for optimal yield and activity.

How can nuclear magnetic resonance (NMR) techniques be applied to study atpE structure?

NMR spectroscopy offers valuable insights into atpE structure and dynamics that complement other structural biology approaches. Strategic application includes:

• Sample preparation considerations:

  • Isotopic labeling (¹³C, ¹⁵N) of recombinant atpE

  • Detergent micelle or lipid nanodisc reconstitution to mimic native membrane environment

  • Concentration optimization to avoid aggregation

• Key NMR experiments:

  • HSQC-TOCSY for resonance assignments

  • NOE measurements for distance constraints

  • Relaxation measurements for dynamics analysis

  • Solid-state NMR for intact c-ring analysis

• Structural analysis focus:

  • Transmembrane helix orientation and packing

  • Proton-binding site conformation

  • Protein-lipid interactions

  • Conformational changes during rotation

NMR data should be integrated with computational modeling and other structural techniques (X-ray crystallography, cryo-EM) for comprehensive structural characterization of this challenging membrane protein complex.

How can the atpE translational enhancer be optimized for heterologous protein expression?

The atpE translational enhancer sequence offers significant potential for improving recombinant protein yields in E. coli. Optimization strategies include:

• Sequence element analysis: Systematic mutation studies of the U-rich and A-rich regions to identify critical nucleotides for enhancement activity. The atpE pattern (UUUUAACUGAAACAAA) can serve as starting template for optimization .

• Positioning optimization: Determining the optimal distance between the enhancer sequence, SD sequence, and start codon for maximum translational efficiency.

• Application to challenging targets: Testing the enhancer with proteins that are typically difficult to express in E. coli, especially those with non-optimal codon usage. Previous studies demonstrate significant enhancement (6-10 fold) even with human genes like IL2 and IFNβ that have codon usage patterns typically associated with low expression in E. coli .

• Combination with other enhancement strategies: Evaluating synergistic effects when combined with codon optimization, chaperone co-expression, or secretion signals.

• Expression vector design: Incorporating the optimized enhancer into a suite of expression vectors with different promoters, fusion tags, and selection markers for versatile application.

This optimization process requires systematic experimental validation with multiple target proteins to establish broadly applicable enhancement parameters.

What are the current contradictions in understanding c-subunit stoichiometry across different organisms?

The stoichiometry of c-subunits in ATP synthase complexes reveals intriguing evolutionary adaptations that remain incompletely understood. Current research contradictions include:

• Variation causality: While c-rings ranging from c₁₀ to c₁₅ have been documented across species, the evolutionary pressures driving this variation remain disputed . Competing hypotheses suggest adaptations to:

  • Environmental pH

  • Energy availability

  • Metabolic requirements

  • Membrane composition

• Methodological inconsistencies: Different structural determination techniques (X-ray crystallography, cryo-EM, AFM, cross-linking) sometimes yield conflicting results for the same organism, raising questions about sample preparation effects on ring integrity.

• Physiological significance: The functional consequence of different coupling ratios (H⁺/ATP ranging from 3.3 to 5.0) remains debated . The energetic advantage of higher efficiency (lower H⁺/ATP) must be balanced against potential disadvantages in certain environmental conditions.

• Regulatory mechanisms: Whether organisms can modulate c-ring composition in response to environmental changes remains controversial, with limited evidence for compositional plasticity in some species.

These contradictions highlight the need for standardized methodologies and comprehensive comparative studies across diverse organisms to resolve fundamental questions about this critical aspect of bioenergetics.

How does ATP synthase function relate to pathogenicity in E. coli O6:K15:H31?

The relationship between ATP synthase function and pathogenicity in E. coli O6:K15:H31 involves several interconnected factors:

• Metabolic adaptation: E. coli O6:K15 is frequently found as both an enterotoxigenic pathogen originally isolated from children with diarrhea and as a uropathogenic strain . The ATP synthase complex likely plays a central role in energy metabolism adaptations required for colonization of these distinct host environments.

• Association with virulence factors: The O6:K15 serotype frequently occurs in combination with the O6 antigen and a mannose-resistant hemagglutinin . This suggests co-evolution of energy metabolism systems (including ATP synthase) with virulence factors.

• Clonal distribution: Orskov et al. demonstrated that certain O and K serotypes frequently occurred together in enteropathogenic E. coli strains isolated from widespread geographic locations, suggesting these strains represent clones adapted to growth in the small intestine . This adaptation likely involves specialized energy metabolism configurations.

• Host response modulation: ATP levels affect numerous cellular processes involved in pathogenesis, including adhesion, invasion, toxin production, and stress response. ATP synthase function may therefore indirectly influence virulence expression.

The specific mechanisms by which ATP synthase modifications might contribute to pathogenicity in this serotype represent an important area for future research, potentially leading to novel therapeutic approaches targeting bacterial bioenergetics.

What experimental approaches can determine the role of atpE in antibiotic resistance mechanisms?

Investigating atpE's potential role in antibiotic resistance requires multifaceted experimental approaches:

• Genetic manipulation studies:

  • Construction of atpE deletion or point mutation strains

  • CRISPR-Cas9 genome editing for precise modifications

  • Complementation studies with wild-type and mutant atpE variants

• Resistance profiling:

  • Minimum inhibitory concentration (MIC) determination for various antibiotic classes

  • Time-kill kinetics to assess bactericidal activity

  • Persistence assays to evaluate survival under antibiotic stress

• Membrane potential analysis:

  • Fluorescent dye-based measurements (DiSC3, TMRM) to assess ΔΨ

  • Correlation between ATP synthase activity and membrane potential during antibiotic exposure

• Metabolic adaptation characterization:

  • Metabolomic profiling to identify shifts in energy metabolism

  • Oxygen consumption and ATP production measurements

  • Gene expression analysis of related pathways

• Structural studies:

  • Analysis of potential antibiotic binding sites on atpE

  • Conformational changes induced by antibiotics that target ATP synthase

  • Competitive binding assays with known ATP synthase inhibitors

These approaches would provide comprehensive insights into how alterations in atpE structure or function might contribute to antibiotic resistance phenotypes in pathogenic E. coli strains.