Recombinant Salmonella dublin ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Salmonella dublin and what is its biological significance?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase in Salmonella dublin. The protein consists of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This highly hydrophobic membrane protein forms the c-ring structure that is essential for proton translocation across the membrane.

The biological significance of atpE lies in its central role in energy production. The c-subunit ring is the rotary component that converts the proton gradient into mechanical energy, which is then used by the F1 sector to synthesize ATP. This process is fundamental to bacterial survival and metabolism, making it an important target for understanding bacterial physiology and developing antimicrobial strategies.

How does ATP synthase c-subunit differ from other components of the ATP synthase complex?

The ATP synthase c-subunit is distinctive in that it forms the membrane-embedded proton-conducting channel of the complex. Unlike the catalytic F1 portion that extends into the cytoplasm, the c-subunit exists primarily within the membrane as part of the F0 sector.

The c-subunit's function is mechanistically different from other components:

• While the F1 sector (containing α, β, γ, δ, and ε subunits) performs the catalytic synthesis of ATP, the c-subunit forms the proton channel

• The c-subunit can form leak channels that regulate inner membrane ATP production efficiency

• The c-subunit has been implicated in the formation of the mitochondrial permeability transition pore (mPTP) in eukaryotic systems

This unique positioning and function make the c-subunit particularly important for understanding energy coupling mechanisms in bacteria.

Why is ATP synthase sometimes incorrectly referred to as ATPase in scientific literature?

The terminology confusion stems from historical experimental limitations. Early research could only measure the ATP hydrolysis (ATPase) activity of isolated components rather than ATP synthesis. As explained in historical accounts from the 1960s and 1970s:

"It is instructive to consider a paper by Kagawa and Racker, published in 1966... they were aiming to understand ATP synthesis, but were at the stage where all they could measure was the ATPase activity of fractions. It was not until 1973 that Racker and Stoeckenius were first able to synthesize ATP..."

The correct terminology distinction is:

• ATP synthase: Uses proton gradient to synthesize ATP from ADP and phosphate

• ATPase: Catalyzes ATP hydrolysis into ADP and phosphate

This distinction is critical for accurate experimental design and interpretation, especially since ATP synthase can work bidirectionally, functioning as an ATPase under certain conditions, such as with excess ATP .

What expression systems are most effective for producing recombinant S. dublin atpE protein?

Based on current research protocols, E. coli expression systems are most commonly used for recombinant production of S. dublin atpE. Commercial recombinant products utilize E. coli as the expression host . The effectiveness of this system stems from:

• Genetic similarity between E. coli and Salmonella dublin

• Well-established protocols for membrane protein expression

• High yield potential with optimized codon usage

For optimal expression, researchers should consider:

This approach mirrors the production method used for commercial recombinant atpE, which utilizes N-terminal His-tagging and E. coli expression systems .

What are the optimal storage and handling conditions for recombinant S. dublin atpE protein preparations?

Proper storage and handling are critical for maintaining the structural integrity and function of recombinant S. dublin atpE. According to established protocols:

• The purified protein should be stored as a lyophilized powder for long-term stability

• Upon reconstitution, the recommended conditions are:

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (optimal: 50%)

  • Store at -20°C/-80°C in small aliquots to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

When handling the protein:

• Briefly centrifuge vials before opening to bring contents to the bottom

• Avoid repeated freeze-thaw cycles as they significantly degrade membrane proteins

• Use Tris/PBS-based buffer with 6% trehalose at pH 8.0 for optimal stability

These conditions maintain protein integrity while preserving the hydrophobic interactions critical for c-subunit structure.

What analytical methods should be employed to confirm the structural integrity of recombinant S. dublin atpE?

Given the challenges of working with highly hydrophobic membrane proteins like atpE, a multi-faceted analytical approach is recommended:

• Electrophoretic analysis:

  • SDS-PAGE with tricine gel systems optimized for small hydrophobic proteins

  • Western blotting using anti-His antibodies (for His-tagged proteins)

• Spectroscopic methods:

  • Circular dichroism (CD) to confirm α-helical secondary structure

  • Fourier-transform infrared spectroscopy (FTIR) to assess membrane protein conformation

• Functional assays:

  • Reconstitution into liposomes to measure proton translocation activity

  • ATP hydrolysis coupling assays when combined with F1 components

• Structural analysis:

  • Mass spectrometry to confirm the intact mass (expected 8.5 kDa) and sequence coverage

  • NMR analysis of detergent-solubilized protein to assess tertiary structure

Minimum purity standards should exceed 90% as determined by SDS-PAGE , with verification of the complete amino acid sequence through proteomic analysis.

How does the ATP synthase c-subunit contribute to Salmonella dublin pathogenicity and host-pathogen interactions?

The ATP synthase c-subunit plays several important roles in S. dublin pathogenicity that extend beyond its primary function in energy metabolism:

• Energy provision for virulence mechanisms:

  • S. dublin is characterized by high invasiveness and antimicrobial resistance (AMR)

  • The ATP synthase provides energy required for invasion, intracellular survival, and AMR efflux pumps

• Potential role in membrane permeability:

  • The c-subunit can form leak channels that affect membrane potential

  • These channels may influence ionic balance and survival in acidic environments of the host

• Contribution to stress adaptation:

  • Research on ATP synthase in Salmonella indicates its involvement in adaptation to environmental stresses encountered during host infection

  • The c-subunit's function may be particularly important during transition from the intestinal environment to systemic invasion

Current research at the University of Edinburgh and Quadram Institute is investigating the genetic factors contributing to S. dublin's invasive nature in both cattle and humans . This research employs cutting-edge genome sequencing and phenotyping techniques to better understand how these factors, potentially including ATP synthase components, facilitate zoonotic transmission and contribute to pathogenicity.

What is known about the regulatory role of ATP synthase c-subunit leak channel in bacterial metabolism?

Recent research has revealed that the ATP synthase c-subunit leak channel plays a significant regulatory role in cellular metabolism:

• Protein synthesis regulation:

  • One key finding is that "the ATP synthase c-subunit leak channel level activity regulates the rate of protein synthesis"

  • This regulation appears to be a mechanism for balancing energy production with cellular metabolic demands

• Efficiency of energy production:

  • The leak that "regulates inner membrane ATP production efficiency resides within the membrane-embedded c-subunit ring of the ATP synthase"

  • This suggests a feedback mechanism where the degree of proton leak through the c-ring modulates the efficiency of ATP production

• Metabolic adaptation:

  • The regulated leak may serve as a mechanism for bacterial adaptation to different energy states

  • By modulating the coupling efficiency between proton translocation and ATP synthesis, bacteria can adjust their energy production to environmental conditions

This regulatory function adds complexity to our understanding of bacterial bioenergetics and suggests potential targets for metabolic manipulation in antimicrobial development.

How does atpE function relate to antimicrobial resistance in Salmonella dublin?

The relationship between ATP synthase c-subunit (atpE) and antimicrobial resistance in S. dublin operates through several mechanisms:

• Energy-dependent resistance mechanisms:

  • Many antibiotic resistance mechanisms, including efflux pumps and enzymatic modifications, require ATP

  • ATP synthase function is therefore indirectly critical for powering these resistance systems

• Membrane potential and drug uptake:

  • The c-subunit's role in maintaining membrane potential affects the uptake of numerous antibiotics

  • Alterations in c-subunit function could modify antibiotic permeability into the cell

• Direct antibiotic targeting:

  • Some antimicrobials directly target ATP synthase (e.g., bedaquiline in Mycobacterium)

  • Mutations in atpE could confer resistance to such ATP synthase-targeting compounds

S. dublin has been identified as particularly concerning due to "its high invasiveness and antimicrobial resistance (AMR)" . Ongoing research funded by the BBSRC is investigating the genetic factors that contribute to these characteristics, which may include adaptations in energy metabolism systems.

How does S. dublin atpE compare structurally and functionally with homologous proteins in other bacterial species?

The ATP synthase c-subunit shows notable conservation across bacterial species while maintaining important structural and functional distinctions:

Functionally, all bacterial c-subunits participate in ATP synthesis through the rotary mechanism, but with some differences:

• Salmonella flagellar systems have evolved a backup engine powered by sodium (Na⁺) motive force which can function when traditional ATP systems are compromised

• The specific structure of the c-ring influences the bioenergetic efficiency, with variations in the number of c-subunits affecting the H⁺/ATP ratio

• Adaptation to different environmental niches has selected for variations in the c-subunit that optimize function under specific conditions (pH, temperature, ion availability)

These comparative insights are valuable for understanding evolutionary adaptations in bacterial bioenergetics and for developing species-specific interventions.

What methodological approaches are most effective for studying protein-protein interactions involving S. dublin atpE?

Due to the membrane-embedded nature of atpE, specialized approaches are required to study its protein-protein interactions:

• Cross-linking methodologies:

  • Chemical cross-linking combined with mass spectrometry

  • Photo-activatable amino acid incorporation at specific positions

  • These approaches can capture transient interactions within the intact ATP synthase complex

• Microscale thermophoresis (MST):

  • Allows detection of interactions in solution with minimal protein amounts

  • Compatible with detergent-solubilized membrane proteins

  • Quantifies binding affinities under near-native conditions

• Förster resonance energy transfer (FRET):

  • Site-specific labeling of atpE and potential interaction partners

  • Enables real-time monitoring of interactions in reconstituted systems

  • Can be adapted for high-throughput screening

• Structural approaches:

  • Cryo-electron microscopy of the intact ATP synthase complex

  • Solid-state NMR of membrane-reconstituted components

  • X-ray crystallography of complexes stabilized by antibody fragments

These methods have been successfully applied to study interactions between c-subunits and other components of the ATP synthase complex, as well as potential interactions with regulatory factors or inhibitors.

What are the key technical challenges in studying the function of S. dublin atpE in vitro?

Researchers face several significant technical challenges when studying S. dublin atpE function:

• Protein solubility and stability:

  • As a highly hydrophobic membrane protein, atpE is difficult to maintain in a stable, functional state outside its native membrane environment

  • Requires careful selection of detergents or lipid reconstitution systems

• Functional reconstitution:

  • Achieving proper orientation and oligomerization of the c-ring structure in artificial membranes

  • Ensuring coupling with other ATP synthase components for functional studies

• Measurement of activity:

  • Direct measurement of proton translocation requires specialized techniques

  • Distinguishing between passive leak and active transport functions

• Structural characterization:

  • Traditional structural biology techniques are challenging with membrane proteins

  • Requires specialized approaches like electron microscopy or solid-state NMR

These challenges can be addressed through:

• Using nanodisc technology for membrane protein stabilization

• Developing coupled enzyme assays for indirect activity measurement

• Employing fluorescent probes sensitive to membrane potential or pH

• Utilizing advanced microscopy techniques for structural characterization

How can researchers leverage S. dublin atpE in developing novel antimicrobial strategies?

The ATP synthase c-subunit presents several promising avenues for antimicrobial development:

• Direct inhibition strategies:

  • Design of small molecules that specifically bind to the c-ring and disrupt rotation

  • Development of peptides that interfere with c-subunit assembly or interaction with other components

  • Targeting of the unique ion channel properties of the c-ring

• Vaccine development approaches:

  • Usage of attenuated S. dublin strains with modified ATP synthase components

  • Current research demonstrates that attenuated S. dublin strains (like Sdu189ΔspiC and Sdu189ΔspiCΔaroA) show potential as live attenuated vaccines

  • These vaccines could potentially incorporate modified atpE to enhance immunity

• Diagnostic applications:

  • Development of detection methods for S. dublin based on atpE sequence variations

  • Creation of antibodies specific to S. dublin atpE for diagnostic testing

The University of Edinburgh and Quadram Institute research aims to "provide insights that will aid the development of effective surveillance, control programmes and potential vaccines" for S. dublin . This work, along with similar initiatives, represents a critical step toward leveraging our understanding of bacterial energy metabolism for public health applications.

What emerging technologies are advancing our understanding of ATP synthase c-subunit function in bacterial pathogens?

Several cutting-edge technologies are revolutionizing research on bacterial ATP synthase c-subunits:

• Single-molecule techniques:

  • High-speed atomic force microscopy allowing visualization of c-ring rotation in real-time

  • Optical tweezers measuring the mechanical forces generated during ATP synthesis

  • These approaches provide unprecedented insights into the mechanics of ATP synthase function

• Advanced structural methods:

  • Cryo-electron microscopy achieving near-atomic resolution of intact ATP synthase

  • Integrative structural biology combining multiple data types for comprehensive models

  • These methods reveal the detailed architecture of the c-ring and its interactions

• Genetic technologies:

  • CRISPR-Cas9 genome editing for precise modification of atpE in its native context

  • High-throughput mutational scanning to map structure-function relationships

  • These approaches enable systematic analysis of atpE variants

• Systems biology approaches:

  • Metabolic flux analysis to determine the impact of atpE modifications on cellular energetics

  • Network analysis integrating transcriptomics, proteomics, and metabolomics data

  • These methods place ATP synthase function within the broader context of bacterial physiology

Current research utilizing "cutting-edge genome sequencing and phenotyping techniques to investigate the genetic factors that contribute to the invasive nature of S. Dublin" exemplifies how these technologies are being applied to understand bacterial pathogens at a systems level.