Recombinant Bacillus halodurans ATP synthase subunit c (atpE)

What is the functional role of ATP synthase subunit c in Bacillus halodurans?

ATP synthase subunit c (atpE) forms the critical rotor component of the F0 domain in the bacterial ATP synthase complex. This subunit creates a ring structure that rotates when protons flow through the complex, directly coupling proton movement to mechanical rotation that drives ATP synthesis. In B. halodurans, as in other bacterial species, subunit c forms the proton-conducting pathway through the membrane, with each c-subunit typically carrying a conserved acidic residue that participates in proton translocation . The rotation of this c-ring ultimately drives conformational changes in the F1 domain, leading to ATP production from ADP and inorganic phosphate.

Unlike some bacterial ATP synthases that exhibit latent ATPase activity, the specific regulatory mechanisms in B. halodurans ATP synthase remain less characterized than those in model organisms like E. coli or other Bacillus species. Research suggests that, similar to other extremophilic bacteria, B. halodurans may possess unique adaptations in its ATP synthase complex to function optimally in alkaline environments.

How does the genomic organization of the atp operon in B. halodurans compare to other Bacillus species?

The atp operon in B. halodurans, like other Bacillus species, contains genes encoding all subunits of the F1F0-ATP synthase complex. While not directly addressed in the available search results for B. halodurans specifically, comparative genomics indicates that bacterial atp operons typically maintain conserved organization across related species. The atpE gene encoding subunit c is generally located within this operon alongside genes for other ATP synthase components.

In the broader genomic context of B. halodurans, we know that certain genes (like oapB) are positioned between essential genes such as map (methionyl aminopeptidase) and infA (translation initiation factor IF-1) . This genomic organization can have implications for gene expression regulation and potential polar effects when attempting genetic manipulations, which researchers should consider when designing knockout or expression studies involving the atp operon.

What expression systems are most effective for producing recombinant B. halodurans atpE?

Based on successful approaches with other Bacillus ATP synthase components, heterologous expression in E. coli represents the most widely used system for producing recombinant bacterial ATP synthase subunits. For instance, researchers have successfully developed "a heterologous expression system in Escherichia coli to produce TA2F1 complexes" from thermoalkaliphilic Bacillus species .

For optimal expression of B. halodurans atpE specifically, consider the following methodological approach:

• Vector selection: pET expression vectors with T7 promoters are typically effective for controlled, high-level expression

• E. coli strain optimization: BL21(DE3) or C41/C43(DE3) strains designed for membrane protein expression

• Expression conditions: Induction at lower temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) to minimize aggregation

• Solubilization strategy: Inclusion of appropriate detergents (e.g., DDM, LDAO) in the lysis buffer, as detergents like lauryldimethylamine oxide have been shown to affect ATP synthase activity

For researchers studying the complete F1F0 complex, co-expression of multiple subunits may be necessary, as demonstrated in studies of other bacterial ATP synthases where recombinant F1 subunits (α, β, δ, and γε) were purified and assembled in vitro .

What are the critical factors for successful purification of functional recombinant atpE?

Purification of functional recombinant atpE requires careful consideration of membrane protein handling techniques:

• Solubilization: The choice of detergent is crucial. For ATP synthase components, mild detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryldimethylamine oxide (LDAO) have proven effective .

• Chromatography strategy: A multi-step approach is typically required:

  • Initial capture via affinity chromatography (His-tag purification)

  • Secondary purification via ion exchange chromatography

  • Final polishing via size exclusion chromatography

• Buffer composition: Including ATP and MgCl2 (typically 2 mM each) in purification buffers helps maintain proper protein conformation and stability, as these cofactors have been shown to be essential for ATP synthase subunit assembly .

• Reconstitution: For functional studies, reconstitution into proteoliposomes may be necessary, especially for proton pumping assays. This typically involves dialysis to remove detergent in the presence of phospholipids.

• Quality assessment: SEC-HPLC can be used to verify proper folding and assembly states, as demonstrated in studies examining subunit interactions in ATP synthase complexes .

The critical challenge remains obtaining sufficient quantities of properly folded protein that retains functionality after purification.

What methods are available for measuring ATP synthase activity in recombinant B. halodurans systems?

Several complementary methods can be employed to assess the functionality of recombinant B. halodurans ATP synthase containing atpE:

• ATP synthesis assay using inverted membrane vesicles:
  This approach directly measures ATP production capacity. As described for other bacterial systems, membrane vesicles are prepared where the ATP synthase orientation allows for ATP synthesis when provided with an artificial proton gradient . The assay typically includes:

  • Preparation of inverted membrane vesicles from cells expressing recombinant ATP synthase

  • Generation of a proton gradient using NADH or succinate

  • Measurement of ATP production using luciferase-based luminescence assays

• ATP hydrolysis assays:
  These measure the reverse reaction (ATP breakdown). For example, in studies with thermoalkaliphilic Bacillus ATP synthase, researchers found that "the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide" . Common approaches include:

  • Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

  • Direct measurement of phosphate release using colorimetric methods

  • Calorimetric approaches measuring heat changes during ATP hydrolysis

• Proton pumping assays:
  These directly measure proton translocation activities:

  • Using pH-sensitive fluorescent dyes (ACMA, pyranine)

  • Measuring pH changes with sensitive electrodes

How can researchers study the assembly of atpE into functional ATP synthase complexes?

Assembly studies are crucial for understanding how atpE integrates into the complete ATP synthase complex. Based on methodologies applied to other bacterial ATP synthases, the following techniques are recommended:

• Size exclusion chromatography (SEC):
  SEC-HPLC has been successfully employed to monitor the assembly of ATP synthase subunits. For example, researchers studying ATP synthase assembly observed that "chromatograms show a shift into a shorter elution peak time confirming αβ heterodimer formation only upon the addition of 2 mM ATP and MgCl2" . This approach can be adapted to monitor atpE incorporation into larger complexes.

• Laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS):
  This emerging technique allows analysis of intact membrane protein complexes and has been used for ATP synthase subunit assembly studies . It can provide information about the stoichiometry and stability of complexes.

• Electron microscopy:
  Cryo-EM or negative staining EM can visualize assembled complexes and confirm proper incorporation of subunits.

• Crosslinking coupled with mass spectrometry:
  This approach can identify interaction interfaces between atpE and other subunits during assembly.

• In vitro reconstitution experiments:
  Similar to approaches where researchers "reconstituted recombinant TA2F1 complexes with F1-stripped native membranes" , reconstitution of purified components can reveal assembly requirements and kinetics.

When conducting assembly studies, it's important to note that ATP and Mg²⁺ (typically at 2 mM concentrations) are often required for proper assembly of ATP synthase components .

What known mutations in atpE affect ATP synthase function and antibiotic resistance?

While specific mutations in B. halodurans atpE have not been extensively characterized in the available search results, important insights can be drawn from studies of ATP synthase in related organisms:

• Antibiotic resistance mutations:
  In S. aureus, a G to T mutation at position 49 in atpE leading to an A17S amino acid substitution confers resistance to tomatidine, an antibiotic that targets the ATP synthase . This suggests that similar positions in B. halodurans atpE might be involved in inhibitor binding.

• Functional domains:
  The c-subunit typically contains a conserved acidic residue (Asp or Glu) that is essential for proton translocation. Mutations of this residue severely compromise ATP synthase function.

• Oligomycin resistance:
  While not specifically mentioned for B. halodurans, mutations conferring resistance to oligomycin (an ATP synthase inhibitor) often map to the c-subunit in various species, providing insights into inhibitor binding sites.

These findings suggest that similar positions in B. halodurans atpE would be prime targets for site-directed mutagenesis studies to elucidate structure-function relationships and potential resistance mechanisms.

What are the current challenges in structural characterization of B. halodurans ATP synthase?

Structural studies of ATP synthase components, particularly the membrane-embedded c-subunit, face several technical challenges:

• Membrane protein crystallization:
  The hydrophobic nature of atpE makes crystallization for X-ray diffraction studies challenging. Alternative approaches may include:

  • Fusion protein strategies to increase solubility

  • Lipidic cubic phase crystallization

  • Nanobody-assisted crystallization

• Cryo-EM considerations:
  While cryo-EM has revolutionized structural studies of ATP synthases, challenges remain:

  • The relatively small size of the c-ring may limit resolution

  • Sample heterogeneity if the complex disassembles during purification

  • Detergent micelle interference with image processing

• NMR approaches:
  Solution NMR of membrane proteins requires:

  • Isotopic labeling strategies (¹³C, ¹⁵N)

  • Optimization of membrane mimetics (nanodiscs, bicelles)

  • Selection of suitable detergents that maintain protein structure

• Computational modeling:
  In lieu of experimental structures, homology modeling based on related bacterial ATP synthases can provide preliminary structural insights. Structural consequences of mutations can be "evaluated in structural models" , as has been done for other ATP synthases.

Researchers should consider the extremophilic nature of B. halodurans when designing structural studies, as this may impact protein stability under experimental conditions.

How do inhibitors target bacterial ATP synthase subunit c, and what is known about selectivity?

ATP synthase inhibitors represent potential antimicrobial agents, with the c-subunit being a validated target. Key insights include:

• Inhibitor mechanisms:
  Compounds like tomatidine and its derivatives target bacterial ATP synthase with high specificity. Research has shown "a correlation between antibiotic potency and ATP synthase inhibition" , with structure-activity relationship studies demonstrating that "the orientation of the position 3 group and an intact spiroaminoketal moiety in tomatidine and its analogs are important for ATP synthase inhibition and whole-cell inhibitory activity" .

• Selectivity metrics:
  Critically for therapeutic applications, high selectivity between bacterial and human mitochondrial ATP synthases is essential. For the tomatidine derivative FC04-100, "the selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10⁵-fold" . This exceptional selectivity makes c-subunit targeting particularly promising.

• Experimental determination of inhibitory activity:
  ATP synthesis inhibition can be measured using membrane vesicle assays. Studies show that various compounds affect ATP synthesis with different potencies:

  • DCCD (N,N′-dicyclohexylcarbodiimide): A classical ATP synthase inhibitor

  • CCCP (cyanide m-chlorophenylhydrazone): Affects the proton gradient

  • Oligomycin: Targets primarily the F₀ sector

  In these assays, IC₅₀ values typically range "from 0.82 ± 0.17 μg/ml to 8.67 ± 1.9 μg/ml for membrane vesicles from S. aureus" , providing benchmarks for comparison with B. halodurans studies.

What methodologies are most effective for screening potential atpE inhibitors?

For researchers developing inhibitors targeting B. halodurans atpE, a multi-tiered screening approach is recommended:

• Biochemical assays:

  • ATP synthesis assays using inverted membrane vesicles provide direct measurement of functional inhibition

  • ATP hydrolysis assays can serve as complementary screens

  • Dose-response curves should be generated to determine IC₅₀ values

• Whole-cell activity testing:

  • MIC (minimum inhibitory concentration) determination

  • Time-kill studies to assess bactericidal versus bacteriostatic effects

  • Studies show clear "correlation between structure, MIC, and IC₅₀" for effective inhibitors

• Selectivity assessment:

  • Parallel testing against bacterial ATP synthase and mitochondrial ATP synthase

  • Calculation of selectivity index (SI) as the ratio of mitochondrial IC₅₀ to bacterial IC₅₀

  • Cytotoxicity testing in mammalian cell lines

• Resistance development monitoring:

  • Serial passage experiments to assess resistance development potential

  • Sequencing of resistant mutants to identify resistance mechanisms

  • Introduction of identified mutations into B. halodurans atpE to confirm the resistance mechanism

• Structural studies:

  • Molecular docking to predict binding modes

  • Structure-activity relationship studies

This comprehensive approach ensures that potential inhibitors are thoroughly characterized before advancing to more resource-intensive studies.

How can recombinant B. halodurans atpE contribute to bioenergetic studies in extremophiles?

B. halodurans is an alkaliphilic bacterium, and its ATP synthase has evolved to function optimally under alkaline conditions. Recombinant atpE from this organism offers unique opportunities for bioenergetic studies:

• Proton motive force paradox investigation:
  Alkaliphiles like B. halodurans face a bioenergetic challenge known as the "proton motive force paradox" - they must synthesize ATP despite a seemingly unfavorable proton gradient. Studies with recombinant atpE can help elucidate:

  • Specialized mechanisms for proton capture

  • Structural adaptations that enhance ATP synthesis efficiency

  • Amino acid substitutions that favor proton binding under alkaline conditions

• Comparative bioenergetic studies:
  By comparing recombinant atpE from B. halodurans with that of neutralophilic bacteria, researchers can gain insights into:

  • Evolutionary adaptations for extreme environments

  • Structural determinants of pH-dependent function

  • Energy conservation strategies in different ecological niches

• Biotechnological applications:
  Understanding the unique properties of B. halodurans ATP synthase could lead to applications such as:

  • Design of pH-resistant bioenergetic systems

  • Development of bioreactors optimized for alkaline conditions

  • Engineering of enzymes with enhanced stability

• Methodological considerations:
  When using recombinant B. halodurans atpE for such studies, researchers should:

  • Ensure experimental conditions (particularly pH) match the native environment

  • Consider the impact of lipid environment on protein function

  • Compare activity across a range of pH values to establish pH-activity profiles

What are the most promising approaches for studying the complete assembly of ATP synthase containing recombinant atpE?

Understanding the complete assembly pathway of ATP synthase with recombinant atpE requires sophisticated experimental approaches:

• Time-resolved assembly studies:
  Similar to approaches used for other bacterial ATP synthases where researchers determined that "assembly of all different subunits follows a specific order" , time-resolved studies can reveal the assembly sequence:

  • Pulse-chase experiments with labeled subunits

  • Time-course studies using size exclusion chromatography

  • Single-molecule techniques to observe assembly events

• Assembly intermediates characterization:
  Studies have shown that "α or β bind to the γ-subunit, whereas the ε-subunit is only weakly bound to γ" , highlighting the importance of characterizing intermediate complexes:

  • Native gel electrophoresis to separate assembly intermediates

  • Mass spectrometry to determine subunit composition

  • Cryo-EM of assembly intermediates

• In vitro reconstitution:
  Complete assembly can be studied through reconstitution experiments:

  • Stepwise addition of purified components

  • Monitoring of activity development during assembly

  • Assessment of the impact of specific conditions (ATP, Mg²⁺, pH) on assembly efficiency

• Genetic approaches for in vivo assembly:

  • Fluorescent protein tagging for visualization of assembly

  • Conditional expression systems to control assembly timing

  • Pull-down assays to identify assembly factors

These approaches provide complementary information about the complex process of ATP synthase assembly, enabling researchers to develop a comprehensive understanding of how atpE is incorporated into the functional complex.

What are the most common issues in recombinant expression of atpE and how can they be addressed?

Membrane proteins like atpE present unique challenges in recombinant expression systems. Common issues and solutions include:

• Low expression levels:

  • Solution: Optimize codon usage for the expression host; try fusion partners such as MBP or SUMO; test different promoter strengths

  • Example: Studies developing expression systems for ATP synthase components have found that controlled expression conditions are critical

• Inclusion body formation:

  • Solution: Lower induction temperature (16-20°C); reduce inducer concentration; use specialized E. coli strains like C41/C43(DE3)

  • Approach: Alternatively, develop refolding protocols from inclusion bodies using gradual detergent introduction

• Toxicity to host cells:

  • Solution: Use tight expression control systems; employ strains with reduced leaky expression

  • Strategy: Consider cell-free expression systems for highly toxic membrane proteins

• Improper membrane insertion:

  • Solution: Include proper signal sequences; co-express appropriate chaperones

  • Consideration: Test different membrane-mimetic environments for proper folding

• Verification of expression:

  • Challenge: Traditional Western blotting may be ineffective if antibodies cannot access epitopes

  • Solution: Include reporter tags at both N- and C-termini; use reporter fusions to verify full-length expression

The development of "a heterologous expression system in Escherichia coli to produce" ATP synthase complexes has been successful for other Bacillus species , suggesting similar approaches could work for B. halodurans atpE.

How can researchers verify the structural integrity and proper folding of purified recombinant atpE?

Verifying the structural integrity of membrane proteins like atpE is crucial but challenging. Recommended approaches include:

• Structural spectroscopy:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • FTIR to assess membrane-embedded structural elements

  • NMR spectroscopy for more detailed structural assessment

• Functional assays:

  • Reconstitution into liposomes followed by proton transport assays

  • Assembly with other ATP synthase components to verify interaction capability

  • Inhibitor binding studies to confirm native conformation

• Protease accessibility:

  • Limited proteolysis to assess folding status

  • Studies with ATP synthase components have shown that properly folded proteins often have distinctive proteolytic patterns, as seen where "the ε subunit was resistant to proteolytic digestion" in its native conformation

• Thermal stability assessment:

  • Differential scanning calorimetry (DSC) to measure unfolding transitions

  • Thermofluor assays to optimize buffer conditions

• Detergent screening:

  • Test multiple detergents to identify those that maintain protein stability

  • Monitor protein behavior in different detergents using size exclusion chromatography

A combination of these approaches provides comprehensive verification of structural integrity, ensuring that purified recombinant atpE represents the native conformation.

What emerging technologies hold promise for advancing B. halodurans atpE research?

Several cutting-edge technologies are poised to transform research on B. halodurans atpE:

• Cryo-EM advances:

  • Developments in sample preparation and image processing allow for higher resolution structures of membrane proteins

  • Time-resolved cryo-EM could potentially capture different conformational states during the catalytic cycle

• Native mass spectrometry:

  • Improvements in membrane protein mass spectrometry techniques enable analysis of intact complexes

  • Can provide insights into subunit stoichiometry and assembly intermediates

• Single-molecule biophysics:

  • FRET-based approaches to study conformational dynamics during rotation

  • Magnetic tweezers or optical traps to directly measure torque generation

• Nanodiscs and other membrane mimetics:

  • Improved systems for maintaining membrane proteins in native-like environments

  • Enable structural and functional studies in the absence of detergents

• AI/ML approaches:

  • AlphaFold and similar tools for improved structural prediction

  • Machine learning for identifying functionally important residues based on sequence analysis

• CRISPR-based genetic tools:

  • Development of genetic systems for direct manipulation of B. halodurans

  • Allows for in vivo study of atpE mutations and their phenotypic effects

These technologies promise to overcome current limitations in studying this challenging but important membrane protein component.

What are the most significant unanswered questions regarding B. halodurans atpE function and regulation?

Despite advances in ATP synthase research, several key questions remain unanswered specifically for B. halodurans atpE:

• Alkaliphilic adaptations:

  • How does the c-subunit in B. halodurans contribute to ATP synthesis under alkaline conditions?

  • What structural features allow proton binding despite unfavorable external pH?

  • Are there unique regulatory mechanisms for controlling ATP synthase activity in alkaliphiles?

• Stoichiometry questions:

  • What is the exact number of c-subunits in the B. halodurans c-ring?

  • How does this stoichiometry compare to non-alkaliphilic bacteria, and what are the functional implications?

• Regulatory interactions:

  • Does B. halodurans employ similar regulatory mechanisms as seen in other bacteria where "the ε subunit acts as an inhibitor of ATPase activity" ?

  • Are there unique protein-protein interactions in the B. halodurans ATP synthase complex?

• Inhibitor interactions:

  • What is the precise binding site for inhibitors like tomatidine derivatives in the c-subunit?

  • Can inhibitors be designed that specifically target B. halodurans-like ATP synthases?

• Evolutionary considerations:

  • How has the extreme environment shaped the evolution of atpE in B. halodurans?

  • What can comparative genomics tell us about the adaptation of ATP synthase to different environmental niches?

Addressing these questions will require integrative approaches combining structural biology, biochemistry, genetics, and computational methods.