Recombinant Anoxybacillus flavithermus ATP synthase subunit c (atpE)

Basic Research Questions

• What is the structure and function of ATP synthase subunit c in Anoxybacillus flavithermus?

  The ATP synthase subunit c (atpE) in A. flavithermus is a highly conserved 75-residue peptide that forms a transmembrane α-helical hairpin structure. Its amino acid sequence is MGVLAAAIAIGLAALGA GIGNGLIVSRTVEGIA RQPEARGMLQTTMFIG VALVEAIPIIA VVIAFMVQGR . This hydrophobic peptide is an integral component of the F₀ complex of ATP synthase, where it assembles into oligomers to form the c-ring that constitutes the main part of the rotor . During ATP synthesis, the c-ring rotates in response to proton flow across the membrane, driving conformational changes in the F₁ sector that catalyze ATP production from ADP and inorganic phosphate .

  The c-subunit plays a crucial role in coupling the proton gradient generated by the respiratory chain to ATP synthesis, directly cooperating with subunit a in the proton pumping process . The protein structure is adapted for function in thermophilic conditions, allowing A. flavithermus to thrive in high-temperature environments .

• How does Anoxybacillus flavithermus adapt to thermophilic environments at the genomic level?

  A. flavithermus strains isolated from geothermal sites on Deception Island in Antarctica have genomes of approximately 3.0 Mb with a G+C content of 42%, encoding about 3500 proteins on average . Genomic analysis reveals specific adaptations for thermophilic growth, including:

  • Genes compatible with growth at high temperatures and alkalophilic conditions (pH > 8.5)

  • Enzymes required for silica adaptation and biofilm formation

  • Proteins for synthesis of long-chain polyamines as components of silica nanospheres

  • Metabolic adaptations for silicification and sinter formation in geothermal environments

  Comparative genome analysis suggests extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli, potentially representing streamlining for specialized thermophilic lifestyles .

• What is the genomic organization of the ATP synthase genes in A. flavithermus?

  The ATP synthase genes in A. flavithermus and related bacteria are typically organized in an operon structure. In Bacillus pseudofirmus OF4, a related alkaliphilic bacterium, the ATP synthase operon contains a small open reading frame upstream of the atpI gene, designated atpZ . This gene encodes a hydrophobic protein with 2 transmembrane helices.

  The atpZ gene appears to be restricted to a specific subset of low %GC Gram-positive bacteria (Firmicutes phylum) and is not found in all members of these genera, suggesting a specialized function potentially related to the physiological setting of these bacteria . The atpE gene encoding subunit c is part of this operon, and disruption of genes in this operon can result in defects in non-fermentative growth, particularly under suboptimal magnesium concentrations .

• How does the ATP synthase of A. flavithermus compare to that of other alkaliphilic bacteria?

  A. flavithermus ATP synthase belongs to the H⁺-coupled type, similar to most aerobic alkaliphilic bacteria. This contrasts with some anaerobic alkaliphiles that use Na⁺-coupled ATP synthases. Below is a comparison table of ATP synthase coupling ions in various alkaliphilic bacteria:

  Aerobes	F-type ATP synthase coupling ion
  Arthrospira platensis	H⁺
  Bacillus alcalophilus	H⁺
  Bacillus clausii	H⁺
  Bacillus halodurans	H⁺
  Bacillus pseudofirmus OF4	H⁺
  Oceanobacillus iheyensis	H⁺
  Anaerobes
  Alkaliphilus metalliredigens	Na⁺
  Alkaliphilus oremlandii	Na⁺
  Bacillus selenitireducens	H⁺
  Natranaerobius thermophilus	Na⁺
  Thioalkalivibrio sp.	H⁺

  H⁺-coupled ATP synthases in alkaliphiles face a bioenergetic challenge, as the protonmotive force (pmf) is reduced at high pH due to the adverse pH gradient (acid inside relative to outside). Unlike some adaptations seen in other alkaliphiles, cyanobacterial Spirulina platensis sequesters its ATP synthase in thylakoids that are not continuous with the cytoplasmic membrane, avoiding the low bulk pmf problem .

• What signal transduction mechanisms are present in A. flavithermus that might influence ATP synthase function?

  A. flavithermus encodes numerous proteins involved in signal transduction, which could potentially regulate energy metabolism and ATP synthase function:

  • 23 sensor histidine kinases and 24 response regulators

  • 20 methyl-accepting chemotaxis proteins

  • 5 predicted eukaryotic-type Ser/Thr protein kinases

  • 21 proteins involved in metabolism of cyclic diguanylate (c-di-GMP)

  The set of c-di-GMP-related proteins is particularly extensive, with A. flavithermus encoding 12 proteins with the diguanylate cyclase (GGDEF) domain, 6 of which also contain the c-di-GMP phosphodiesterase (EAL) domain, and one combining GGDEF with an alternative c-di-GMP phosphodiesterase (HD-GYP) domain . C-di-GMP is a secondary messenger that regulates transition from motility to sessility and biofilm formation, which may be relevant to the organism's survival in thermal environments .

Advanced Research Questions

• What experimental challenges are associated with expressing and purifying recombinant A. flavithermus atpE?

  Working with recombinant A. flavithermus ATP synthase subunit c presents several experimental challenges:

  • The protein is highly hydrophobic, requiring specialized handling techniques

  • Proper storage is critical: recommended at -20°C/-80°C with 50% glycerol as stabilizer

  • Shelf life is approximately 6 months for liquid form and 12 months for lyophilized form at -20°C/-80°C

  • Reconstitution protocol requires precise conditions: deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol

  • Repeated freezing and thawing should be avoided; working aliquots should be stored at 4°C for up to one week

  • As a membrane protein, functional studies may require reconstitution into liposomes or nanodiscs to maintain native structure

  Expression in E. coli has been reported , but optimizing conditions for high-yield expression of properly folded protein likely requires screening different expression systems, detergents, and purification strategies.

• How does calcium influence the structure and function of ATP synthase subunit c?

  Calcium has profound effects on ATP synthase subunit c structure and function, as demonstrated in studies of the mammalian c subunit:

  • Ca²⁺ can induce conformational changes in the c-subunit, causing it to transition from α-helical to β-sheet structure

  • The c-subunit is an amyloidogenic peptide that can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a Ca²⁺-dependent manner

  • In the absence of Ca²⁺, minimal amyloid formation occurs, while adding 1 mM Ca²⁺ significantly accelerates formation of β-sheet structures as measured by Thioflavin T fluorescence

  • Ca²⁺ appears to favor formation of oligomers rather than fibrils, which may be relevant to membrane permeabilization mechanisms

  • These Ca²⁺-induced structural changes may contribute to mitochondrial pathology through participation in calcium-induced permeability transition

  While these studies were performed on mammalian c-subunit, the high conservation of this protein suggests similar structural transitions might occur in the bacterial homolog, though this requires experimental verification.

• What methodologies are most effective for studying structural transitions of ATP synthase subunit c?

  Several complementary approaches have proven effective for studying the structural properties and transitions of ATP synthase subunit c:

  • Thioflavin T (ThT) assay: Monitors formation of β-sheet aggregates over time; sample preparation involves incubating 5 μM protein with 30 μM ThT at 37°C with or without 1 mM Ca²⁺, measuring fluorescence at 30-second intervals (excitation 440 nm, emission 480 nm)

  • Dot blot analysis: Detects specific conformations using conformation-specific antibodies; freshly prepared samples are incubated with or without Ca²⁺ at 37°C for 4 hours, spotted on PVDF membrane, and probed with appropriate antibodies

  • Atomic force microscopy: Provides direct visualization of oligomers and fibrils

  • Black lipid membrane methods: Assess ion channel activity of reconstituted protein

  • Circular dichroism spectroscopy: Monitors secondary structure transitions between α-helical and β-sheet conformations

  • Fluorescence spectroscopy: Can track structural changes using intrinsic or extrinsic fluorophores

  These methods, used in combination, can provide comprehensive insights into the structural dynamics of the c-subunit under different conditions.

• What is the role of ATP synthase subunit c in microbial thermotolerance and heat shock response?

  ATP synthase c-subunit likely plays an important role in thermotolerance in Anoxybacillus flavithermus, which can survive at temperatures up to 100°C . Several aspects of this relationship can be investigated:

  • Structural stability of the c-ring at high temperatures is critical for maintaining ATP synthesis

  • Potential changes in proton binding and release kinetics at elevated temperatures

  • Role in maintaining membrane integrity and proton impermeability at high temperatures

  • Possible involvement in spore formation and heat resistance

  Experimental approaches to study this relationship include:

  • Heat shock experiments (e.g., exposure to 75°C for 20 minutes) to assess survival and recovery

  • Comparative analysis of c-subunit sequences and structures across thermophiles with different temperature optima

  • Site-directed mutagenesis to identify residues critical for thermostability

  • Measurement of ATP synthase activity at different temperatures

  Understanding these mechanisms could provide insights into both fundamental aspects of thermophile biology and potential biotechnological applications.

• How does the proton coupling mechanism work in A. flavithermus ATP synthase at high pH and temperature?

  The ATP synthesis reaction catalyzed by ATP synthase can be represented as:

  ADP + Pi + nH⁺(P-side) ⟶ ATP + H₂O + nH⁺(N-side)

  Where n is the number of protons translocated per ATP synthesized, and P-side and N-side represent the electrically positive and negative sides of the membrane, respectively .

  In alkaliphiles like A. flavithermus, ATP synthesis at high pH presents a thermodynamic challenge due to:

  • Reduced availability of substrate H⁺ at high cytoplasmic pH

  • Adverse pH gradient (acid inside relative to outside) that reduces the protonmotive force

  • Need to maintain proton coupling despite these challenges

  Unlike some alkaliphiles that use Na⁺-coupled ATP synthases to overcome this problem, A. flavithermus maintains H⁺-coupling . Possible adaptive mechanisms include:

  • Specialized structural features of the c-ring that optimize proton binding/release at high pH

  • Altered stoichiometry of protons translocated per ATP synthesized

  • Delocalized proton gradients near the membrane surface

  • Membrane-associated microcircuits between H⁺ pumping complexes and synthases

  • Proximity-dependent coupling between respiratory chain components and ATP synthase

  These adaptations likely depend on specific membrane properties and alkaliphile-specific modifications of both the ATP synthase and respiratory chain components .

• How can inhibitors be used to study the function of A. flavithermus ATP synthase subunit c?

  Several inhibitors can be employed to study ATP synthase function and potentially identify unique features of the A. flavithermus enzyme:

  • Dicyclohexylcarbodiimide (DCCD): Covalently modifies the conserved carboxyl group in the c-subunit that is essential for proton transport

  • Oligomycin: Binds to the interface between subunit a and the c-ring, blocking proton translocation

  • 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl): Modifies nucleotide binding sites

  • Sodium azide (NaN₃): General inhibitor of the F₁ portion

  • Metal fluoride complexes: AlFₓ, ScFₓ, and BeFₓ can trap different conformational states of the enzyme

  • Polyphenols: Natural compounds like resveratrol, piceatannol, quercetin, morin, and epicatechin can inhibit ATP synthase function

  Experimental approaches using these inhibitors include:

  • Concentration-dependent inhibition assays to determine IC₅₀ values

  • Comparison of inhibition profiles between A. flavithermus and mesophilic ATP synthases

  • Structure-activity relationship studies to identify inhibitor binding determinants

  • Molecular docking and dynamics simulations to predict inhibitor binding modes

  • Site-directed mutagenesis to validate predicted inhibitor binding sites

  These studies can provide valuable insights into the unique structural and functional properties of A. flavithermus ATP synthase.

• What are the potential biotechnological applications of A. flavithermus ATP synthase subunit c?

  The thermostable ATP synthase components from A. flavithermus have several potential biotechnological applications:

  • Bioenergy research: Development of thermostable ATP-producing systems for biofuel cells

  • Nanobiotechnology: Creation of nanomachines based on the rotary mechanism of ATP synthase

  • Drug discovery: Use as a model system for developing antimicrobials targeting bacterial ATP synthases

  • Structural biology: Thermostable proteins often crystallize more readily, facilitating structural studies

  • Protein engineering: Basis for creating chimeric or modified ATP synthases with novel properties

  • Biosensors: Development of ATP-sensing devices that function at elevated temperatures

  The unique properties of proteins from extremophiles like A. flavithermus make them valuable templates for protein engineering and biotechnological innovation . Their stability under harsh conditions makes them particularly suitable for industrial applications requiring robust enzymatic activities.

• How do silica adaptation mechanisms in A. flavithermus relate to ATP synthase function?

  A. flavithermus has been found in super-saturated silica solutions and in opaline silica sinter, with specific adaptations for silica environments:

  • Genome sequencing has identified enzymes required for silica adaptation and biofilm formation

  • Proteomic analysis confirmed regulation of biofilm-related proteins and enzymes for synthesis of long-chain polyamines (constituents of silica nanospheres)

  • ATP synthesis must be maintained during silicification and sinter formation

  The relationship between silica adaptation and ATP synthase function may involve:

  • Protection of the ATP synthase complex during silicification

  • Maintenance of proton gradients in silica-rich environments

  • Potential structural modifications of ATP synthase components for stability in silica matrices

  • Integration of energy metabolism with biofilm formation in silica-containing thermal habitats

  Studying these relationships requires approaches such as:

  • Comparative proteomic analysis of A. flavithermus grown with and without silica

  • Measurement of ATP synthase activity in silica-containing media

  • Electron microscopy studies of cell envelope and ATP synthase localization during silicification

  • Analysis of gene expression patterns in silica-rich environments

  This research could provide insights into both bacterial adaptation mechanisms and biomineralization processes relevant to early Earth environments .

• How can structural biology approaches be applied to study A. flavithermus ATP synthase subunit c?

  Several structural biology approaches can be applied to study the A. flavithermus ATP synthase c-subunit:

  • X-ray crystallography: Requires purification and crystallization of the c-ring or complete ATP synthase complex

  • Cryo-electron microscopy: Particularly suitable for membrane protein complexes; can visualize ATP synthase within native-like environments

  • Solid-state NMR: Can provide atomic-resolution information on membrane proteins in lipid bilayers

  • Molecular dynamics simulations: Can predict structural dynamics and proton transport mechanisms based on atomic models

  • Cross-linking mass spectrometry: Identifies protein-protein interactions within the ATP synthase complex

  • Hydrogen-deuterium exchange mass spectrometry: Maps structural dynamics and solvent accessibility

  Experimental considerations specific to thermophilic proteins include:

  • Temperature-dependent structural studies to identify thermally induced conformational changes

  • Comparison with mesophilic homologs to identify determinants of thermostability

  • Analysis in different lipid environments to mimic native thermal habitats

  • Investigation of pH-dependent structural changes to understand alkaliphilic adaptations

  Structural insights could help resolve fundamental questions about ATP synthesis mechanisms in extremophiles and potentially inspire biomimetic energy conversion technologies.

• What insights can comparative genomics provide about ATP synthase evolution in thermophilic bacteria?

  Comparative genomics of ATP synthase genes across thermophilic bacteria can reveal:

  • Evolutionary adaptations to high temperature and pH environments

  • Horizontal gene transfer events that may have contributed to extremophile adaptation

  • Conserved residues critical for function versus those that vary to accommodate different environments

  • The relationship between genome size, gene content, and metabolic efficiency in thermophiles

  A. flavithermus has undergone extensive gene loss after divergence from other bacilli, suggesting streamlining for its specialized lifestyle . Analysis of ATP synthase genes in the context of this evolutionary history could reveal:

  • Selection pressures on ATP synthase genes in different thermal environments

  • Co-evolution of ATP synthase components with other cellular systems

  • Potential acquisition of specialized genes (like atpZ) that may modulate ATP synthase function

  • Evidence for convergent evolution in ATP synthases from unrelated thermophiles

  Research approaches include:

  • Phylogenetic analysis of ATP synthase genes across diverse thermophiles

  • Detection of signatures of positive selection in ATP synthase sequences

  • Analysis of genomic context and operon organization across species

  • Reconstruction of ancestral sequences to track evolutionary trajectories

  This comparative approach can provide a broader context for understanding how ATP synthases adapt to extreme conditions.