Recombinant Geobacillus thermodenitrificans ATP synthase subunit delta (atpH)

What is the role of the delta subunit in bacterial ATP synthases?

The delta subunit (atpH) in bacterial ATP synthases is part of the F1 domain, which represents the extrinsic membrane portion of the ATP synthase complex. It plays a crucial role in the structural stability of the enzyme by connecting the F1 catalytic domain to the Fo membrane domain. Unlike its minimal role in some bacterial systems, the delta subunit in thermophilic bacteria like G. thermodenitrificans is likely critical for maintaining enzyme stability at elevated temperatures. The delta subunit contributes to the function of the enzyme not by direct catalysis but by maintaining the proper structural arrangement required for efficient ATP synthesis and hydrolysis .

Why is G. thermodenitrificans a preferred thermophilic expression system?

G. thermodenitrificans has emerged as a valuable host for expression of thermostable proteins due to several key advantages:

• High transformation efficiency: Optimized electroporation procedures can achieve efficiencies of 10³ to 10⁵ CFU/μg for various plasmid types .

• Growth at elevated temperatures: As a thermophile, it grows optimally at high temperatures, allowing for screening of thermostable protein variants.

• Protein production capability: G. thermodenitrificans efficiently produces heterologous proteins in both intracellular and extracellular environments .

• Genetic tractability: The availability of deletion mutants (e.g., ΔresA) with enhanced transformation efficiency makes genetic manipulation more feasible .

These characteristics make G. thermodenitrificans particularly suitable for expressing thermostable proteins like ATP synthase components that require proper folding at elevated temperatures.

What expression vectors are compatible with G. thermodenitrificans for recombinant atpH production?

For recombinant expression in G. thermodenitrificans, several plasmid types have been demonstrated to work with varying efficiencies. Research has shown that:

• Plasmids prepared from dam mutant E. coli strains are accepted with significantly higher efficiency compared to those from dam+ strains, suggesting the circumvention of a restriction-modification system .

• Multiple compatible plasmids with different copy numbers and segregational stabilities can be used simultaneously in G. thermodenitrificans K1041 .

• The ΔresA mutant strain exhibits transformation efficiencies of >10⁵ CFU/μg for some plasmids, making it particularly suitable for difficult-to-transform constructs .

When choosing an expression vector for atpH production, researchers should consider temperature-stable selection markers and promoters that function efficiently at elevated temperatures.

How can I optimize expression conditions for functional recombinant G. thermodenitrificans atpH protein?

Optimizing expression of functional G. thermodenitrificans atpH requires careful consideration of several parameters:

Expression System Selection:

• Homologous expression in G. thermodenitrificans: Provides proper folding environment at high temperatures

• Heterologous expression in E. coli: Easier genetic manipulation but may require refolding

Critical Optimization Parameters:

When expressing in E. coli, the Tuner(DE3) strain has been successfully used for expressing thermostable enzymes from G. thermodenitrificans, as demonstrated with the esterase Est1 . For ATP synthase components, co-expression with molecular chaperones may improve folding and solubility, especially when expressing at temperatures below the protein's physiological environment.

What purification strategy yields the highest recovery of functional atpH protein?

Purification of recombinant G. thermodenitrificans atpH requires a specialized approach due to its thermostable nature and potential integration into the ATP synthase complex:

Recommended Purification Workflow:

• Heat treatment: Exploit the thermostability of G. thermodenitrificans proteins by heating crude lysate (65-70°C for 15-20 minutes) to precipitate mesophilic host proteins while keeping the target protein in solution.

• Immobilized metal affinity chromatography (IMAC): For His-tagged atpH constructs, use Ni-NTA columns with gradual imidazole elution (50-300 mM).

• Ion exchange chromatography: Based on the theoretical pI of atpH, select appropriate ion exchange media for further purification.

• Size exclusion chromatography: Final polishing step to obtain homogeneous protein preparations.

Buffer Optimization:

This strategy typically yields 2-5 mg of purified protein per liter of bacterial culture with >90% purity as assessed by SDS-PAGE and Western blotting .

How can I assess the functional integration of recombinant atpH into the ATP synthase complex?

Functional integration assessment requires multiple complementary approaches:

• ATP synthesis/hydrolysis assays: Measure ATP synthesis rates in reconstituted liposomes or ATP hydrolysis activity using the standard malachite green phosphate detection method. For ATP synthesis, use membrane vesicles prepared from cells expressing the recombinant protein and generate a proton gradient using NADH or an acid-base transition .

• Subunit interaction analysis:

  • Pull-down assays using tagged atpH to identify binding partners

  • Surface plasmon resonance to measure binding kinetics with other ATP synthase subunits

  • Cross-linking followed by mass spectrometry to map interaction interfaces

• Structural validation:

  • Limited proteolysis to assess proper folding

  • Circular dichroism spectroscopy to evaluate secondary structure elements

  • Thermal shift assays to determine stability changes compared to wild-type

Functional reconstitution tests have shown that in some bacteria, truncated versions of ATP synthase components can still participate in ATP synthesis. For example, in the thermophilic bacterium Bacillus PS3, even severely truncated γ subunits (γQ36stop and γP43stop) were able to catalyze ATP synthesis at 25-35% of wild-type rates , suggesting flexibility in structural requirements for minimal ATP synthase function.

What structural features distinguish G. thermodenitrificans atpH from mesophilic bacterial delta subunits?

The structural adaptations in G. thermodenitrificans atpH that confer thermostability likely include:

• Increased ionic interactions: Higher proportion of salt bridges stabilizing tertiary structure

• Enhanced hydrophobic core: More extensive burial of hydrophobic residues

• Reduced loop regions: Shorter, more rigid loops connecting secondary structure elements

• Higher proline content: Increased proline residues in loop regions providing conformational rigidity

Comparative sequence analysis indicates that thermophilic ATP synthase delta subunits typically contain 5-10% more charged residues (Arg, Lys, Glu, Asp) than their mesophilic counterparts, facilitating stabilizing ionic interactions. Additionally, thermophilic variants often display a higher aliphatic index, reflecting the increased hydrophobicity of buried residues that contributes to protein stability at elevated temperatures .

Structural studies of bacterial ATP synthases have revealed that the delta subunit serves as an important connector between the F₁ and F₀ domains. In Bacillus PS3, a thermophilic relative of G. thermodenitrificans, the ATP synthase has been structurally characterized, showing how this simple bacterial enzyme performs the same core functions as more complex mitochondrial ATP synthases .

How does atpH contribute to the regulatory mechanisms of ATP synthesis/hydrolysis in thermophilic bacteria?

The delta subunit plays a specialized role in the regulatory mechanisms of thermophilic bacterial ATP synthases:

• Structural stabilization: By bridging the F₁ and F₀ domains, atpH helps maintain the proper structural alignment necessary for efficient rotational catalysis, especially critical under thermal stress .

• Regulation of inhibitory mechanisms: In bacterial ATP synthases, several inhibitory mechanisms prevent wasteful ATP hydrolysis when proton motive force (pmf) collapses. Though inhibition is primarily mediated by other subunits (ε in many bacteria), the delta subunit's positioning affects how these regulatory elements interact with the catalytic core .

• Thermal adaptation of rotational mechanics: The delta subunit in thermophilic bacteria must maintain precise structural connections despite thermal motion, requiring specific adaptations in its interaction surfaces with other subunits .

Research on bacterial ATP synthases has demonstrated species-specific differences in ATP hydrolysis inhibition. For instance, in Bacillus subtilis, ATP hydrolysis is significantly inhibited compared to other bacterial F₁F₀ complexes, with the strength of inhibition correlating with ADP(Mg²⁺) occupancy in the catalytic sites . These regulatory differences likely extend to the interactions of the delta subunit with other components of the complex.

What experimental approaches can resolve conflicting data on atpH subunit orientation in the ATP synthase complex?

Resolving conflicting structural data on atpH orientation requires multi-faceted approaches:

Complementary Structural Techniques:

• Cryo-electron microscopy (cryo-EM):

  • Single-particle analysis of purified ATP synthase complexes

  • Sub-classification of particles to identify conformational heterogeneity

  • Focus on resolving the F₁-F₀ interface where atpH is located

• Cross-linking mass spectrometry (XL-MS):

  • Use zero-length or short-length cross-linkers to capture native interactions

  • Map cross-linked residues to identify proximity relationships

  • Compare cross-linking patterns under different energetic states

• Site-directed spin labeling combined with EPR spectroscopy:

  • Introduce spin labels at key residues in atpH

  • Measure distances between labeled sites in different conditions

  • Monitor orientational changes during catalytic cycle

Validation Through Functional Studies:

Bacterial ATP synthases have been successfully imaged using cryo-EM, as demonstrated for the Bacillus PS3 ATP synthase expressed in E. coli, allowing atomic models to be built in different rotational states . Such approaches can be adapted to specifically investigate the orientation and dynamics of the delta subunit in G. thermodenitrificans ATP synthase.

How can G. thermodenitrificans atpH be utilized as a model for understanding ATP synthase evolution?

G. thermodenitrificans atpH serves as an excellent model for evolutionary studies due to several factors:

• Thermophilic adaptation: Comparing atpH sequences across thermophilic and mesophilic bacteria provides insights into thermal adaptation mechanisms of essential cellular machinery.

• Evolutionary intermediary: Positioned between simple bacterial and complex eukaryotic ATP synthases, G. thermodenitrificans ATP synthase components can illuminate evolutionary transitions in this ancient molecular motor.

• Minimal functional requirements: Studies of bacterial ATP synthases have revealed surprising functional resilience despite structural simplifications. For instance, research has shown that the γ subunit of Bacillus PS3 ATP synthase can be reduced to just its N-terminal helix while still supporting ATP synthesis, suggesting evolutionary insights into the minimal structural requirements for function .

Comparative genomic analyses indicate that while the core function of ATP synthase is conserved across domains of life, the regulatory mechanisms have diversified significantly. G. thermodenitrificans atpH may represent an evolutionary adaptation that balances efficiency and regulation in high-temperature environments.

What methodological approaches are most effective for studying atpH interactions with other ATP synthase subunits?

Multiple complementary methods are required to comprehensively map atpH interactions:

In vitro Interaction Studies:

• Isothermal titration calorimetry (ITC): Quantify binding thermodynamics between purified atpH and partner subunits

• Surface plasmon resonance (SPR): Measure association/dissociation kinetics of subunit interactions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map interaction interfaces by identifying regions protected from exchange

In vivo Interaction Validation:

• Bacterial two-hybrid assays: Adapted for thermophilic conditions to test interactions

• Co-immunoprecipitation: Using antibodies against atpH or epitope tags

• FRET-based approaches: Using fluorescent protein fusions expressed in G. thermodenitrificans

Computational Interaction Prediction:

Studies of bacterial ATP synthases, including thermal stability observations, suggest that the delta subunit's interactions are critical for maintaining the association between F₁ and F₀ domains. In thermophilic bacteria like Bacillus PS3, these interactions must be particularly stable to maintain function at elevated temperatures .

How can structural insights from atpH research contribute to developing novel antimicrobial strategies?

Structural knowledge of G. thermodenitrificans atpH can inform antimicrobial development through several avenues:

• Identification of species-specific regulatory elements: The F₁F₀-ATP synthase is essential for bacterial viability and has been validated as a drug target, particularly in mycobacteria . Structural studies have revealed that species-specific elements, including the C-terminal domain of the α subunit and unique features of the γ and δ subunits, are critical for function and represent attractive targets for selective inhibition .

• Understanding thermal adaptation mechanisms: By comparing thermophilic and mesophilic ATP synthases, researchers can identify critical structural differences that could be exploited for selective targeting of pathogenic bacteria.

• Designing inhibitors of subunit assembly: Disrupting the assembly of ATP synthase components by targeting the interaction interfaces of atpH with other subunits represents a novel antimicrobial strategy.

Research has demonstrated that mycobacterium-specific elements of the α, γ, and δ subunits are attractive targets for species-specific inhibitors . Similar approaches could be applied to other bacterial pathogens by leveraging the structural knowledge gained from studying thermophilic ATP synthases.

What strategies can overcome poor expression yields of recombinant G. thermodenitrificans atpH?

Poor expression yields of recombinant atpH can be addressed through systematic optimization:

Expression System Refinement:

• Codon optimization: Adjust codon usage to match the expression host

• Expression vector selection: Test promoters with different strengths and induction mechanisms

• Host strain engineering: Use strains with enhanced capacity for thermostable protein expression

Expression Condition Optimization:

Solubility Enhancement:

• Fusion tags: Test solubility-enhancing fusion partners (MBP, SUMO, TrxA)

• Buffer optimization: Screen various buffer compositions with stabilizing additives

For thermophilic proteins expressed in mesophilic hosts, heat shock prior to induction can pre-condition the cellular machinery for better handling of thermostable proteins. Additionally, the use of E. coli Tuner(DE3) strain has proven successful for expressing other thermostable enzymes from G. thermodenitrificans .

How can researchers troubleshoot non-functional recombinant atpH in ATP synthase activity assays?

When recombinant atpH fails to show functionality in ATP synthase assays, systematic troubleshooting is required:

Protein Quality Assessment:

• Verify structural integrity: Circular dichroism spectroscopy to confirm secondary structure

• Check thermal stability: Differential scanning calorimetry to assess if the protein maintains its expected thermostability

• Evaluate oligomeric state: Size exclusion chromatography to determine if proper oligomerization occurs

Functional Assay Troubleshooting:

• Reconstitution conditions: Optimize lipid composition and protein-to-lipid ratios for membrane reconstitution

• Assay buffer composition: Ensure proper ion concentrations, particularly Mg²⁺ which is critical for ATP synthase function

• Energization methods: Test alternative methods to generate proton motive force

Molecular Level Diagnostics:

Research on ATP synthases has shown that even truncated versions of some subunits can support function. For example, in Bacillus PS3 ATP synthase, constructs with severely truncated γ subunits (γQ36stop and γP43stop) were able to catalyze ATP synthesis at 25-35% of wild-type rates . This suggests that when troubleshooting functionality, testing minimal constructs focused on core interaction domains may be informative.

What are the critical controls needed when investigating atpH-mediated effects on ATP synthase regulation?

Rigorous controls are essential when studying atpH's regulatory functions:

Essential Experimental Controls:

• Negative controls:

  • ATP synthase complex lacking atpH to establish baseline activity

  • Catalytically inactive mutants (e.g., βE190Q) to distinguish enzyme-dependent effects

  • Heat-denatured samples to control for non-enzymatic reactions

• Positive controls:

  • Wild-type ATP synthase complex for activity benchmarking

  • Known regulatory conditions (e.g., ADP-inhibition) to validate assay sensitivity

  • Step-wise reconstitution with defined subunit composition

• Specificity controls:

  • Point mutations in predicted interaction interfaces

  • Chimeric atpH constructs with domains from related species

  • Competition assays with excess free atpH

Critical Parameters to Monitor:

When studying ATP hydrolysis inhibition mechanisms, it's important to note that different bacterial species show varying degrees of inhibition. For instance, ATP hydrolysis is significantly inhibited in Bacillus subtilis and very strongly inhibited in P. denitrificans compared to other bacterial ATP synthases . These differences should be considered when interpreting the effects of atpH modifications on regulatory functions.

How might CRISPR-Cas9 genome editing in G. thermodenitrificans advance atpH functional studies?

CRISPR-Cas9 genome editing offers transformative approaches for studying atpH function:

• Precise genomic modifications:

  • Introduction of point mutations to test structure-function hypotheses

  • Creation of deletion variants to assess domain contributions

  • Insertion of epitope tags for tracking endogenous protein

• Regulatory element manipulation:

  • Promoter replacements to control expression levels

  • Introduction of inducible systems for temporal regulation

  • Engineering of reporter fusions for activity monitoring

• High-throughput functional genomics:

  • Implementation of CRISPR interference (CRISPRi) for conditional knockdowns

  • Creation of saturating mutation libraries for comprehensive functional mapping

  • Combinatorial modifications of multiple ATP synthase components

The high transformation efficiency achieved in G. thermodenitrificans K1041, particularly in the ΔresA mutant strain , provides an excellent foundation for CRISPR-based approaches. Additionally, the thermostability of Cas9 variants from thermophilic organisms could enhance genome editing efficiency at the elevated growth temperatures of G. thermodenitrificans.

What emerging structural biology techniques show promise for capturing atpH dynamics during the ATP synthesis cycle?

Several cutting-edge structural techniques offer new insights into atpH dynamics:

• Time-resolved cryo-EM:

  • Captures structural snapshots during the catalytic cycle

  • Microfluidic mixing devices allow millisecond time resolution

  • Classification algorithms can sort particles by conformational state

• Single-molecule FRET spectroscopy:

  • Monitors distance changes between labeled residues in real-time

  • Can detect transient conformational states

  • Operates under physiologically relevant conditions

• Integrative structural approaches:

Cryo-EM has already been successfully applied to bacterial ATP synthases, revealing the structure of Bacillus PS3 ATP synthase in three rotational states . These approaches could be extended to specifically track the movements of the delta subunit during the catalytic cycle, potentially revealing its role in coordinating rotational catalysis at high temperatures.

How can systems biology approaches integrate atpH research into broader understanding of bioenergetics in thermophilic bacteria?

Systems biology offers holistic frameworks for understanding atpH's role in thermophilic bioenergetics:

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and metabolomics data

  • Track changes in ATP synthase components under different growth conditions

  • Identify co-regulated genes and proteins in bioenergetic pathways

• Metabolic modeling:

  • Integrate ATP synthase function into genome-scale metabolic models

  • Predict energetic consequences of atpH modifications

  • Simulate growth under different environmental conditions

• Network analysis of protein-protein interactions:

  • Map the extended interaction network of ATP synthase components

  • Identify regulatory proteins that interact with atpH

  • Discover novel bioenergetic pathways unique to thermophilic bacteria

The draft genomic sequence of G. thermodenitrificans K1041 contains 3,384 coding genes , providing a foundation for systems-level analyses. By integrating atpH research into this broader genomic context, researchers can gain insights into how ATP synthase function is coordinated with other cellular processes in thermophilic environments.