Recombinant Bacillus amyloliquefaciens ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c in Bacillus species?

ATP synthase subunit c (atpE) in Bacillus species forms part of the Fo domain of the FoF1-ATP synthase complex. This protein consists of two α-helices connected by a short loop and arranges in a symmetrical oligomeric ring structure (c-ring) within the membrane . In Bacillus PS3, the c-ring typically contains 10 c-subunits . The primary function of subunit c is to participate in proton translocation through Fo, which drives rotation of the c-ring relative to the a-subunit . This rotational motion is mechanically coupled to ATP synthesis in the F1 domain.

Key structural features include:

• Two transmembrane α-helices connected by a hydrophilic loop

• A critical glutamic acid residue (E56 in Bacillus PS3, equivalent to E61 in mycobacterial species) that is essential for proton binding and transport

• A highly conserved amino acid sequence across species, particularly in regions involved in proton translocation

How does the c-ring of ATP synthase contribute to energy conversion in Bacillus species?

The c-ring functions as a proton-driven turbine that converts the electrochemical gradient across the membrane into mechanical energy. When protons bind to the key glutamic acid residue (E56 in Bacillus PS3) in subunit c, they induce conformational changes that facilitate rotation of the c-ring . This rotation is then transmitted to the central stalk of the F1 domain, inducing conformational changes in the catalytic β subunits that lead to ATP synthesis.

Recent research has demonstrated that this process involves cooperation among c-subunits within the ring . Studies with Bacillus PS3 ATP synthase have shown that:

• Proton binding and release events in different c-subunits are coordinated

• The rotation occurs in discrete steps, with each step corresponding to the movement of one c-subunit past the a-subunit

• The energy transduction is inherently cooperative across the c-ring structure

What are the most effective methods for recombinant expression of Bacillus amyloliquefaciens atpE in E. coli systems?

For successful recombinant expression of B. amyloliquefaciens atpE in E. coli, researchers should consider the following methodological approach:

• Vector selection: A bicistronic expression system has shown success in expressing similar membrane proteins . Consider vectors with tightly controlled promoters like pBAD or pET systems.

• Host strain optimization: E. coli BL21(DE3)pLysS has demonstrated effectiveness for membrane protein expression due to its reduced protease activity and controlled expression levels .

• Expression conditions:

  • Temperature: Lower temperatures (16-25°C) typically yield better results for membrane proteins

  • Induction: Mild induction with lower concentrations of inducers (0.1-0.5 mM IPTG for pET systems or 0.002-0.02% arabinose for pBAD)

  • Media: Enriched media like Terrific Broth supplemented with 1% glucose during growth phase

• Membrane integration approach: For proper folding and integration of atpE into membranes:

  • Co-expression with chaperones like DnaK/DnaJ

  • Addition of membrane-stabilizing agents (glycerol 5-10%)

  • Use of E. coli C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression

• Bioreactor cultivation: For scale-up production, both batch and fed-batch DO-stat controlled strategies have shown effectiveness .

What purification strategies maximize yield and stability of recombinant atpE protein?

The purification of recombinant atpE requires specialized approaches due to its hydrophobic nature and membrane localization:

• Membrane isolation:

  • Cell disruption by sonication or French press in buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl₂, and protease inhibitors

  • Differential centrifugation: low-speed centrifugation (10,000 × g) to remove cell debris followed by ultracentrifugation (150,000 × g) to pellet membranes

• Detergent solubilization:

  • Mild detergents like n-dodecyl-β-D-maltoside (DDM) at 1-2% or digitonin at 1% are recommended

  • Solubilization should be performed at 4°C for 1-2 hours with gentle agitation

• Affinity purification:

  • His-tagged constructs can be purified using Ni-NTA resins

  • Washing with increasing imidazole concentrations (10-40 mM) to remove non-specific binding

  • Elution with 250-300 mM imidazole

• Size exclusion chromatography:

  • Further purification using Superdex 200 columns

  • Buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.02-0.05% DDM

• Stability enhancement:

  • Addition of phospholipids (0.02-0.05 mg/ml) such as E. coli polar lipids

  • Addition of glycerol (10%) for long-term storage

  • Storage at -80°C after flash-freezing in liquid nitrogen

How do specific mutations in the atpE gene affect ATP synthase function in Bacillus species?

Mutations in key residues of the atpE gene can significantly impact ATP synthase function. Based on studies with Bacillus PS3 and comparative analysis with mycobacterial atpE, we can identify several critical residues:

• E56 (Bacillus PS3): Equivalent to E61 in mycobacteria; this glutamic acid residue is essential for proton binding and transport. The E56D mutation reduces ATP synthesis and proton pump activities without completely eliminating them, while E56Q abolishes these activities completely . This demonstrates that:

  • The presence of a carboxyl group is critical for proton translocation

  • The precise positioning of this carboxyl group affects efficiency

• A63 (equivalent position in mycobacteria): Mutations at this position (e.g., A63P in M. tuberculosis) affect the α-helix near the critical glutamic acid residue, altering drug binding and potentially affecting proton translocation .

• I66 (equivalent position in mycobacteria): The I66M mutation introduces steric hindrance at the surface of the α-helix near the proton-binding site .

*Based on equivalent positions in mycobacterial atpE, which shares high sequence conservation with Bacillus species.

How does distance between mutations in the c-ring affect cooperative interactions in ATP synthase?

Research on Bacillus PS3 ATP synthase has demonstrated that the spatial arrangement of mutations within the c-ring significantly affects function through cooperative interactions . Using a genetically fused single-chain c-ring containing 10 c-subunits, researchers examined ATP synthase with various combinations of wild-type and E56D-mutated c-subunits.

Key findings include:

• A single E56D mutation substantially decreases ATP synthesis activity but does not eliminate it .

• Double E56D mutations further reduce activity, with the degree of reduction dependent on the spatial arrangement of the mutations .

• Activity decreases progressively as the distance between two mutation sites increases . For example, in the double mutants designated "ef," "eg," "eh," "ei," and "ej," where the letters indicate the position of the E56D mutations, activity decreased as the mutations were placed further apart within the c-ring.

• Molecular dynamics simulations revealed that the mechanism involves sharing of prolonged proton uptake times between mutated c-subunits . As the distance between mutations increases, this sharing effect decreases, explaining the observed activity patterns.

This demonstrates that c-subunits do not function independently but exhibit cooperative behavior in the c-ring, with the spatial arrangement of residues critical for optimal function.

How conserved is the atpE sequence across different Bacillus species compared to other bacterial genera?

The atpE gene shows high conservation across bacterial species, reflecting the essential nature of ATP synthase in cellular energetics. Comparative analysis of atpE sequences reveals:

• Within Bacillus genus: The atpE gene shows very high conservation among Bacillus species, with typically >90% nucleotide identity and >95% amino acid identity .

• Between Bacillus and other genera: Based on comparisons with mycobacterial species, we can estimate that Bacillus atpE shares approximately 70-80% nucleotide identity with more distant bacterial genera .

• Conservation of key functional residues: The region involved in proton translocation, particularly the critical glutamic acid residue and surrounding structure, is highly conserved across nearly all species . Notable exceptions include Mycobacterium xenopi, which has a natural substitution at position 63 (Ala to Met) that may confer natural resistance to certain inhibitors .

What structural adaptations in Bacillus atpE contribute to its thermal stability compared to mesophilic bacteria?

Bacillus species, particularly thermophilic strains like Bacillus PS3, have evolved specific structural adaptations in atpE that enhance thermal stability while maintaining function:

• Amino acid composition: Thermophilic Bacillus species typically show:

  • Increased proportion of charged residues forming salt bridges

  • Higher content of hydrophobic residues in the core regions

  • Decreased occurrence of thermolabile residues (Asn, Gln, Cys, Met)

• α-helical packing: The two α-helices in thermophilic Bacillus atpE exhibit tighter packing through optimized complementary surfaces and enhanced van der Waals interactions .

• Ionic interactions: Strategic positioning of charged residues allows for formation of additional salt bridges that stabilize the structure at higher temperatures.

• Proton-binding site adaptations: The microenvironment around the critical glutamic acid residue (E56 in Bacillus PS3) is modified to maintain appropriate pKa values at elevated temperatures .

• Lipid interactions: The outer surface of the c-ring in thermophilic Bacillus species shows adaptations for optimal interaction with membrane lipids that have higher melting temperatures.

These adaptations collectively contribute to the remarkable thermal stability of ATP synthase in thermophilic Bacillus species while preserving the fundamental mechanism of energy conversion.

What methodologies can accurately measure c-ring rotation in Bacillus ATP synthase for functional studies?

Advanced biophysical techniques have been developed to directly observe and quantify c-ring rotation in ATP synthase. For researchers working with Bacillus ATP synthase, the following methodologies are recommended:

• Single-molecule fluorescence resonance energy transfer (smFRET):

  • Attach donor fluorophore to one c-subunit and acceptor fluorophore to the stator

  • Use total internal reflection fluorescence (TIRF) microscopy for improved signal-to-noise ratio

  • Analyze energy transfer efficiency changes that correspond to rotational movement

  • Temporal resolution: 1-10 ms; spatial resolution: 1-5 nm

• Gold nanorod attachment and dark-field microscopy:

  • Attach gold nanorods (40×10 nm) to the c-ring

  • Observe rotation through polarized light scattering

  • Calculate rotational velocity and step size from intensity fluctuations

  • Temporal resolution: <1 ms; spatial resolution: <1°

• Magnetic bead rotation assays:

  • Attach magnetic beads (200-500 nm) to the c-ring

  • Apply weak magnetic field and track rotation using high-speed video microscopy

  • Calculate torque and step size from rotational behavior

  • Temporal resolution: 0.5-5 ms; angular resolution: 2-5°

• Reconstitution of ATP synthase into liposomes for functional assays:

  • Reconstituate purified ATP synthase into liposomes with defined lipid composition

  • Establish proton gradient using valinomycin/K+ or acid-base transition

  • Measure ATP synthesis rate under defined ΔpH and Δψ conditions

  • Correlate with structural data to assess rotation efficiency

How can genetic engineering approaches be used to create thermostable variants of B. amyloliquefaciens atpE for biotechnological applications?

Creating thermostable variants of B. amyloliquefaciens atpE requires systematic genetic engineering approaches based on structural insights and evolutionary principles:

• Rational design based on comparative analysis:

  • Align atpE sequences from B. amyloliquefaciens with thermophilic Bacillus species (e.g., Bacillus PS3)

  • Identify key residues that differ between mesophilic and thermophilic species

  • Introduce mutations that mimic the thermophilic pattern while preserving function

  • Focus particularly on surface residues that contribute to inter-subunit interactions

• Directed evolution approach:

  • Create a library of random atpE mutants using error-prone PCR

  • Express in an appropriate host system (E. coli BL21(DE3)pLysS with pBAD or similar vector)

  • Screen for functional ATP synthase activity at progressively increasing temperatures

  • Validate promising candidates through purification and in vitro assays

• Chimeric constructs:

  • Create fusion proteins combining domains from thermophilic and mesophilic species

  • Test functionality using ATP synthesis assays in reconstituted systems

  • Analyze structural integrity using circular dichroism and thermal shift assays

• Disulfide bond engineering:

  • Identify positions suitable for introducing cysteine pairs that could form stabilizing disulfide bonds

  • Create single and multiple disulfide variants

  • Test thermal stability while verifying that functional rotation is not impaired

• Computational design and validation:

  • Use molecular dynamics simulations to predict stability of engineered variants at different temperatures

  • Calculate free energy changes (ΔΔG) for stabilizing mutations

  • Implement promising mutations and validate experimentally

The most successful approach typically combines multiple strategies, starting with rational design based on comparative analysis, followed by directed evolution to fine-tune the engineered variants.

How is atpE expression regulated in Bacillus species under different environmental stresses?

In Bacillus species, atpE expression is regulated as part of the ATP synthase operon in response to various environmental stresses:

• Acid stress response:

  • Bacillus species have evolved mechanisms to maintain intracellular pH homeostasis under acid stress

  • ATP synthase expression is modulated to adjust proton extrusion capacity

  • The SigB-dependent general stress response regulates ATP synthase components under acid stress conditions

  • Acid adaptation can involve changes in ATP synthase activity to maintain PMF

• Nutrient limitation:

  • Carbon catabolite repression (CCR) mediated by CcpA affects ATP synthase expression

  • CcpA binding to cre sites in the promoter region can repress or activate genes based on carbon availability

  • CcpA regulates hundreds of genes during different growth phases, potentially including ATP synthase components

• Growth phase regulation:

  • Expression levels of ATP synthase components change during transition from exponential to stationary phase

  • This regulation involves global regulators like CcpA and alternative sigma factors

• Post-translational regulation:

  • By analogy with mycobacterial systems, protein phosphorylation may regulate ATP synthase activity

  • In M. tuberculosis, protein tyrosine phosphatase (PtpA) regulates ATP synthase alpha subunit

  • Similar regulatory mechanisms may exist in Bacillus species, though direct evidence is still emerging

What is the relationship between ATP synthase subunit c expression and biofilm formation in Bacillus species?

The relationship between ATP synthase subunit c (atpE) expression and biofilm formation involves complex energy metabolism adaptations:

• Energy requirements during biofilm development:

  • Biofilm formation requires significant energy investment during initial attachment and matrix production

  • ATP synthase activity is critical for providing this energy, with potential upregulation during early biofilm stages

  • As biofilms mature, central metabolism shifts, potentially affecting ATP synthase expression patterns

• pH regulation in biofilms:

  • Biofilms often develop pH microenvironments due to metabolic activities

  • ATP synthase helps maintain intracellular pH homeostasis under these conditions

  • The pH gradient across the membrane is both used by ATP synthase and affected by its activity

• Coordination with stress responses:

  • Biofilm formation is often triggered by stress conditions

  • General stress response regulators like SigB affect both biofilm formation and ATP synthase regulation

  • This suggests coordinated regulation through common regulatory networks

• Metabolic heterogeneity in biofilms:

  • Cells in different biofilm regions experience different nutrient and oxygen availability

  • This may lead to differential expression and activity of ATP synthase throughout the biofilm structure

  • Spatial regulation of energy metabolism supports the complex architecture and resilience of biofilms

While direct evidence linking atpE expression specifically to biofilm formation in Bacillus species is still emerging, the fundamental connection between energy metabolism and biofilm development suggests this is an important relationship warranting further investigation.

What are the most promising approaches for developing selective inhibitors of bacterial ATP synthase that target the c-subunit?

The development of selective inhibitors targeting bacterial ATP synthase c-subunit represents an important research direction:

• Structure-based drug design:

  • Utilize high-resolution structures of bacterial c-rings to identify unique binding pockets

  • Focus on regions that differ between bacterial and mammalian c-subunits

  • Molecular docking studies to screen virtual libraries for potential binding molecules

  • Refinement through medicinal chemistry approaches

• Lessons from existing inhibitors:

  • Diarylquinolines like R207910 (bedaquiline) target mycobacterial ATP synthase c-subunit

  • Resistance mutations (A63P, I66M) identify critical interaction points

  • Structural models of inhibitor binding can guide development of novel compounds

  • Cross-resistance profiles help understand binding site characteristics

• Species specificity considerations:

  • Natural resistance of M. xenopi to R207910 correlates with A63M substitution

  • Comparative analysis of c-subunit sequences can identify species-specific targetable differences

  • Design of narrow-spectrum inhibitors based on specific structural features

• Alternative approaches:

  • Peptide inhibitors mimicking critical interfaces within the ATP synthase complex

  • Allosteric inhibitors affecting c-ring rotation rather than directly blocking the proton channel

  • Compounds disrupting c-subunit oligomerization during biogenesis

  • Covalent inhibitors targeting accessible, non-conserved cysteine residues

How might synthetic biology approaches utilize engineered ATP synthase c-subunits to create novel energy-harvesting systems?

Synthetic biology offers exciting possibilities for engineering ATP synthase c-subunits for novel applications:

• Reverse engineering for synthetic energy production:

  • Creation of hybrid ATP synthases with engineered c-rings optimized for specific applications

  • Development of artificial proton gradients powered by light or chemical energy

  • Integration with other biological systems to create self-sustaining energy modules

• Nanoscale rotary motors:

  • Engineering c-rings with modified proton binding sites to alter rotation characteristics

  • Creation of molecular motors with controllable speed and torque

  • Integration into nanomechanical devices for targeted applications

• Sensors and switches:

  • Engineering c-subunits to respond to specific environmental signals

  • Development of ATP synthase-based biosensors that convert detection events into measurable ATP production

  • Creation of biological switches that respond to pH, voltage, or chemical gradients

• Novel substrate specificity:

  • Engineering c-subunits to transport ions other than protons (e.g., sodium, potassium)

  • Creation of ATP synthases powered by alternative ion gradients

  • Development of systems optimized for specific environmental conditions

• Biocompatible power sources:

  • Integration of engineered ATP synthases into artificial cell systems

  • Development of implantable energy-generating systems for medical applications

  • Creation of self-sustaining bioreactors for continuous ATP production