Recombinant Bacillus weihenstephanensis ATP synthase subunit c (atpE)

What is Bacillus weihenstephanensis ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the ATP synthase complex in Bacillus weihenstephanensis. It functions as part of the membrane-embedded proton channel that drives ATP synthesis. In B. weihenstephanensis strain KBAB4, this protein has 72 amino acids with a highly hydrophobic profile suitable for its membrane-spanning role. It is also known by alternative names including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, and lipid-binding protein, with the gene being designated as atpE (BcerKBAB4_5108) .

How does the amino acid sequence of B. weihenstephanensis atpE compare with other Bacillus species?

The amino acid sequence of B. weihenstephanensis atpE (MSLGVIAAAIAIGLSALGAGIGNGLIVSRTIEGVARQPELKGALQTIMFIGVALVEALPIIGVVIAFIVMNK) shows high conservation in the hydrophobic regions that form the membrane-spanning domains. Comparative analysis with other Bacillus species reveals conserved functional motifs, particularly the ion-binding site that is critical for proton translocation. Species-specific variations typically occur in the connecting loop regions, potentially reflecting adaptations to different environmental conditions or energy requirements .

What is the phylogenetic relationship between B. weihenstephanensis and other members of the Bacillus cereus group?

B. weihenstephanensis belongs to the Bacillus cereus group (Bcg), which includes several closely related species such as B. cereus, B. thuringiensis, B. anthracis, and the recently characterized B. shihchuchen. Phylogenetic analysis based on conserved proteins, including ATP synthase components, positions B. weihenstephanensis as a psychrotolerant (cold-tolerant) member of this group. Recent genomic studies indicate that B. weihenstephanensis shares significant genomic similarity with certain B. thuringiensis strains, with average nucleotide identity often exceeding 95% .

What are the optimal storage conditions for recombinant B. weihenstephanensis atpE protein?

For optimal stability of recombinant B. weihenstephanensis atpE protein, store at -20°C for regular use or -80°C for extended storage periods. The protein is typically stored in a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein. For working experiments, prepare small aliquots and store at 4°C for up to one week to minimize protein degradation. Importantly, repeated freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity and activity .

How can I optimize the expression and purification of recombinant B. weihenstephanensis atpE?

For optimal expression of recombinant B. weihenstephanensis atpE:

• Vector selection: Choose expression vectors with strong, inducible promoters compatible with the host system (typically E. coli BL21(DE3) or similar strains)

• Codon optimization: Consider codon optimization for the expression host to improve translation efficiency

• Expression conditions:

  • Induce at OD600 0.6-0.8 with IPTG (0.1-0.5 mM)

  • Lower induction temperature (16-25°C) for membrane proteins

  • Extended expression time (12-16 hours) at lower temperatures

For purification:

• Membrane fraction isolation using ultracentrifugation (100,000 × g)

• Solubilization with mild detergents (DDM, LDAO, or C12E8)

• Affinity chromatography using His-tag or other fusion tags

• Size exclusion chromatography for final polishing

This approach addresses the common challenges associated with membrane protein expression, including protein aggregation and inclusion body formation .

What methods are recommended for functional characterization of ATP synthase subunit c?

For comprehensive functional characterization of ATP synthase subunit c, a multi-method approach is recommended:

• Proton translocation assays:

  • ACMA fluorescence quenching to measure proton pumping activity

  • Pyranine-based intravesicular pH measurements

• ATP synthesis/hydrolysis measurements:

  • Luciferin-luciferase bioluminescence assay for ATP synthesis

  • Malachite green assay for quantifying released inorganic phosphate

• Structural studies:

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

  • NMR spectroscopy for atomic-level structural information in membrane mimetics

• Oligomerization analysis:

  • Blue native PAGE to assess c-ring formation

  • Cross-linking assays followed by SDS-PAGE

• Inhibitor binding studies:

  • Isothermal titration calorimetry with specific F0 inhibitors

  • Competitive binding assays with radioactively labeled inhibitors

These methods provide complementary information about the functional integrity and mechanistic properties of the recombinant protein .

What is the predicted structure of B. weihenstephanensis ATP synthase subunit c and how does it relate to function?

The B. weihenstephanensis ATP synthase subunit c is predicted to adopt a hairpin-like structure with two transmembrane α-helices connected by a short hydrophilic loop. Key structural features include:

• N-terminal transmembrane helix (residues 4-31): Contains conserved polar residues that participate in proton binding

• Cytoplasmic loop (residues 32-39): Contains charged residues that interact with other F0 subunits

• C-terminal transmembrane helix (residues 40-67): Contains the critical proton-binding glutamate residue

These subunits assemble into a circular ring (c-ring) with 8-15 copies depending on the species, forming the rotary element of the ATP synthase. The proton-binding site is located at the interface between adjacent c-subunits, with a conserved glutamate residue serving as the proton acceptor/donor. The sequential protonation and deprotonation of these sites drives the rotation of the c-ring, which is mechanically coupled to the catalytic F1 sector to drive ATP synthesis .

How does the atpE protein interact with other subunits in the ATP synthase complex?

The atpE protein (subunit c) forms multiple critical interactions within the ATP synthase complex:

• c-c interactions: Each c-subunit interacts with neighboring c-subunits through hydrophobic residues to form the c-ring structure.

• c-a interactions: The outer surface of the c-ring interfaces with subunit a, forming the proton channel. This interaction occurs via specific residues in the transmembrane helices and is essential for proton translocation.

• c-b interactions: The N-terminal domain of subunit b makes contact with the cytoplasmic side of the c-ring, contributing to the stability of the entire complex.

• c-ε/γ interactions: The c-ring makes contact with the central stalk (γ and ε subunits) of the F1 sector, forming the rotary coupling that drives ATP synthesis.

These interactions are dynamic during catalysis, with the c-ring rotating against the relatively stationary a-subunit. Mutations at these interaction interfaces can significantly impact complex assembly, stability, and catalytic efficiency .

What role does the B. weihenstephanensis atpE play in bacterial energy metabolism?

The B. weihenstephanensis atpE plays a central role in bacterial energy metabolism through several mechanisms:

• Oxidative phosphorylation: It forms the proton-conducting channel in the F0 sector of ATP synthase, utilizing the proton motive force to generate ATP.

• Maintenance of PMF: Under certain conditions, ATP synthase can operate in reverse, hydrolyzing ATP to pump protons and maintain the proton motive force.

• Adaptation to low temperature: As a psychrotolerant bacterium, B. weihenstephanensis has evolved specific adaptations in its ATP synthase to function efficiently at lower temperatures, likely including modifications in the c-subunit to maintain appropriate flexibility and proton conductance.

• Stress response: During environmental stress, the regulation of ATP synthase activity, including the c-subunit, helps bacteria balance energy production and conservation.

The highly conserved nature of this protein across bacterial species underscores its fundamental importance in cellular bioenergetics and survival .

How does B. weihenstephanensis atpE differ from homologous proteins in other Bacillus species?

Comparative analysis of B. weihenstephanensis atpE with homologs from other Bacillus species reveals several key differences:

The differences primarily occur in the transmembrane regions and at the interfacial residues between subunits. These variations likely reflect adaptations to different ecological niches and environmental conditions, particularly the cold tolerance characteristic of B. weihenstephanensis .

What is the significance of B. weihenstephanensis in bacterial phage research?

B. weihenstephanensis has emerged as an important model in bacteriophage research for several reasons:

• Phage susceptibility: B. weihenstephanensis strains show susceptibility to various phages, including those that can also infect other members of the Bacillus cereus group.

• Cross-species activity: Studies have demonstrated that certain phage-derived enzymes, such as LysEFR-4, can effectively lyse B. weihenstephanensis cells, indicating shared cell wall characteristics with related Bacillus species.

• Phage resistance mechanisms: Analysis of B. weihenstephanensis genomes has revealed various phage resistance mechanisms, including CRISPR-Cas systems and restriction-modification systems, providing insights into bacteria-phage co-evolution.

• Therapeutic applications: The susceptibility of B. weihenstephanensis to specific phages and phage-derived enzymes suggests potential applications in biocontrol strategies against pathogenic Bacillus species .

How is B. weihenstephanensis related to pathogenicity in the Bacillus cereus group?

While B. weihenstephanensis itself is generally considered less pathogenic than other members of the Bacillus cereus group, research indicates important connections to pathogenicity:

• Genetic relationships: B. weihenstephanensis shares significant genetic similarity with pathogenic members of the B. cereus group, including recently characterized species like B. shihchuchen biovar anthracis, which contains virulence factors such as anthrax toxin components.

• Virulence factor distribution: Comparative genomic analyses reveal that some B. weihenstephanensis strains may carry genes encoding potential virulence factors, including enterotoxins and hemolysins, albeit often with different expression patterns.

• Environmental reservoir: As a soil bacterium, B. weihenstephanensis may serve as a reservoir for virulence genes that can be horizontally transferred to more pathogenic species.

• Antimicrobial resistance: Some B. weihenstephanensis strains harbor antibiotic resistance genes similar to those found in B. shihchuchen biovar anthracis, including beta-lactamases and efflux pumps, which may contribute to the spread of resistance in environmental and clinical settings .

How can recombinant B. weihenstephanensis atpE be used in structural biology studies?

Recombinant B. weihenstephanensis atpE offers several valuable applications in structural biology:

• Cryo-EM studies: The c-ring formed by multiple atpE subunits is amenable to high-resolution structural analysis by cryo-electron microscopy, particularly when stabilized with appropriate lipids and detergents.

• X-ray crystallography: Purified atpE proteins can be crystallized, either individually or as part of the c-ring assembly, to determine atomic-resolution structures.

• NMR spectroscopy: The relatively small size of atpE makes it suitable for solution and solid-state NMR studies, which can provide detailed information about protein dynamics in membrane environments.

• Molecular dynamics simulations: Structural data from experimental methods can inform computational models to study conformational changes during proton translocation.

• Structure-based drug design: High-resolution structures of atpE can facilitate the development of specific inhibitors targeting the ATP synthase of pathogenic Bacillus species.

These structural studies can reveal critical insights into the mechanisms of energy transduction and provide templates for the design of antimicrobial compounds .

What role does recombinant B. weihenstephanensis atpE play in investigating bacterial adaptation to environmental conditions?

Recombinant B. weihenstephanensis atpE serves as an excellent model for studying bacterial adaptation:

• Cold adaptation mechanisms: As B. weihenstephanensis is psychrotolerant, its atpE can be studied to understand adaptations that maintain membrane fluidity and protein function at low temperatures (4-7°C).

• pH adaptation: Mutations in atpE can alter proton binding affinity, providing insights into how bacteria adapt to different pH environments.

• Energy efficiency: Comparative studies with atpE from mesophilic Bacillus species can reveal adaptations that optimize energy conversion efficiency under different growth conditions.

• Stress response: The regulation and modification of atpE under various stress conditions (temperature, pH, salt) can elucidate stress adaptation mechanisms.

• Environmental niche specialization: The specific properties of B. weihenstephanensis atpE may reflect adaptations to its ecological niche, providing insights into evolutionary processes driving bacterial specialization .

How can site-directed mutagenesis of B. weihenstephanensis atpE advance our understanding of ATP synthase mechanisms?

Site-directed mutagenesis of B. weihenstephanensis atpE provides a powerful approach to investigate ATP synthase function:

• Proton-binding site analysis:

  • Mutating the conserved glutamate residue involved in proton binding

  • Altering surrounding residues to understand the proton-binding microenvironment

• Subunit interface studies:

  • Modifying residues at the c-c subunit interfaces to study c-ring assembly

  • Changing residues at the a-c interface to investigate proton translocation pathways

• Coupling mechanism investigation:

  • Introducing mutations at the interface with the central stalk to study mechanical coupling

  • Altering the flexibility of transmembrane helices to examine conformational changes

• Inhibitor binding studies:

  • Mutating residues known to interact with ATP synthase inhibitors to identify binding determinants

  • Creating inhibitor-resistant variants to validate drug targets

• Temperature adaptation research:

  • Introducing mutations that mimic mesophilic homologs to identify cold-adaptation determinants

  • Creating chimeric proteins with domains from thermophilic bacteria

Results from these studies can provide mechanistic insights into ATP synthase function and guide the development of antimicrobials targeting specific bacterial species .

What are common challenges in working with recombinant B. weihenstephanensis atpE and how can they be addressed?

Researchers working with recombinant B. weihenstephanensis atpE often encounter several challenges:

• Expression issues:

  • Challenge: Low expression yields due to toxicity to host cells

  • Solution: Use tightly controlled expression systems; lower induction temperature to 16-20°C; consider cell-free expression systems

• Protein solubility:

  • Challenge: Formation of inclusion bodies

  • Solution: Express with solubility tags (MBP, SUMO); optimize detergent selection for solubilization; consider native chemical ligation of synthetic peptides

• Purification difficulties:

  • Challenge: Co-purification of host cell membrane proteins

  • Solution: Implement multiple purification steps; use ion exchange chromatography after initial affinity purification; consider density gradient centrifugation

• Functional reconstitution:

  • Challenge: Loss of activity during reconstitution into liposomes

  • Solution: Optimize lipid composition to mimic bacterial membranes; use gentle reconstitution methods; verify proper orientation in liposomes

• Structural heterogeneity:

  • Challenge: Conformational heterogeneity interfering with structural studies

  • Solution: Use stabilizing mutations; employ conformation-specific antibodies; consider nanodiscs for homogeneous membrane environments .

How can I verify the structural integrity of purified recombinant B. weihenstephanensis atpE?

To verify the structural integrity of purified recombinant B. weihenstephanensis atpE, employ a multi-method approach:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to confirm α-helical secondary structure (expected spectrum with minima at 208 and 222 nm)

  • Tryptophan fluorescence to assess tertiary structure integrity

  • Thermal stability assays to determine melting temperature

• Biochemical verification:

  • Size exclusion chromatography to confirm monodispersity

  • Native PAGE to assess oligomeric state

  • Limited proteolysis to verify proper folding (correctly folded proteins show distinct proteolytic patterns)

• Functional assays:

  • Reconstitution into liposomes followed by proton translocation assays

  • ATP synthesis activity when combined with F1 sector components

  • Inhibitor binding studies using fluorescence quenching or isothermal titration calorimetry

• Structural validation:

  • Negative-stain electron microscopy to visualize c-ring formation

  • Crosslinking experiments to verify predicted proximity relationships

  • Mass spectrometry to confirm correct post-translational modifications

These complementary approaches provide comprehensive validation of protein structural integrity prior to downstream applications .

What are the considerations for designing immunological studies using B. weihenstephanensis atpE?

When designing immunological studies with B. weihenstephanensis atpE, researchers should consider:

• Epitope selection:

  • Target the hydrophilic loop region (residues 32-39) as it is surface-exposed

  • Consider species-specific regions to develop discriminating antibodies

  • Analyze potential cross-reactivity with homologous proteins in related species

• Antibody production strategy:

  • Use synthetic peptides corresponding to hydrophilic regions for immunization

  • Consider KLH or BSA conjugation to enhance immunogenicity

  • Implement a prime-boost immunization schedule for higher affinity antibodies

• Validation protocols:

  • Perform Western blot analysis against purified protein and whole-cell lysates

  • Include appropriate controls (pre-immune serum, related Bacillus species)

  • Validate specificity using knockout strains or heterologous expression systems

• Applications optimization:

  • For immunoprecipitation: optimize detergent conditions to maintain native structure

  • For immunohistochemistry: optimize fixation methods to preserve epitope accessibility

  • For ELISA: determine optimal coating conditions and blocking agents

• Cross-reactivity considerations:

  • Test against related Bacillus species to assess specificity

  • Evaluate potential for detecting contamination in environmental or food samples

  • Consider pre-absorption steps if cross-reactivity is observed

These considerations ensure the development of robust immunological tools for studying B. weihenstephanensis atpE in various research contexts .

What are emerging applications of B. weihenstephanensis atpE in biotechnology?

Emerging applications of B. weihenstephanensis atpE in biotechnology include:

• Bioenergy applications:

  • Engineering modified ATP synthases with enhanced efficiency for biotechnological ATP production

  • Developing hybrid systems combining photosynthetic components with bacterial ATP synthases

• Biosensor development:

  • Creating ATP synthase-based biosensors for detecting environmental toxicants that disrupt membrane potential

  • Developing proton flux sensors using modified atpE proteins

• Antimicrobial development:

  • Using the structure of atpE to design specific inhibitors against pathogenic Bacillus species

  • Developing phage-derived lysins that target cell membranes containing ATP synthase complexes

• Nanotechnology applications:

  • Harnessing the rotary motion of ATP synthase for nanomachines and molecular motors

  • Creating biohybrid materials incorporating functional ATP synthase components

• Synthetic biology platforms:

  • Engineering minimal cells with optimized ATP synthase systems

  • Developing oscillating energy systems for dynamic control of synthetic circuits

These applications leverage the unique properties of B. weihenstephanensis atpE, particularly its cold-adaptation features and structural stability .

How might research on B. weihenstephanensis atpE contribute to understanding bacterial pathogenesis?

Research on B. weihenstephanensis atpE can advance our understanding of bacterial pathogenesis through several avenues:

• Evolutionary relationships:

  • Comparative analysis with atpE from pathogenic species can reveal evolutionary adaptations

  • Studying horizontal gene transfer patterns involving ATP synthase components

• Virulence regulation:

  • Investigating links between energy metabolism and virulence factor expression

  • Understanding how ATP synthase activity modulates pathogen adaptation to host environments

• Host-pathogen interactions:

  • Examining how bacterial ATP synthase components interact with host immune responses

  • Investigating potential recognition of bacterial ATP synthase components by host pattern recognition receptors

• Antibiotic resistance connections:

  • Exploring relationships between energy metabolism and antibiotic tolerance

  • Studying how membrane potential, influenced by ATP synthase activity, affects drug uptake

• Novel therapeutic targets:

  • Developing ATP synthase inhibitors specific to pathogenic Bacillus species

  • Investigating synergistic effects between ATP synthase inhibitors and conventional antibiotics

These research directions could provide valuable insights into pathogenesis mechanisms within the Bacillus cereus group and guide the development of novel therapeutic strategies .

What computational approaches can advance our understanding of B. weihenstephanensis atpE function?

Advanced computational approaches offer powerful tools for studying B. weihenstephanensis atpE:

• Molecular dynamics simulations:

  • All-atom simulations in explicit membrane environments to study conformational dynamics

  • Coarse-grained simulations to investigate c-ring assembly and rotation

  • Free energy calculations to quantify proton translocation energetics

• Quantum mechanical/molecular mechanical (QM/MM) methods:

  • Hybrid calculations to study proton transfer mechanisms at atomic resolution

  • Electronic structure analysis of the proton binding site

• Systems biology approaches:

  • Flux balance analysis incorporating ATP synthase activity parameters

  • Whole-cell modeling to predict phenotypic effects of atpE modifications

• Artificial intelligence applications:

  • Machine learning for predicting function-altering mutations

  • Deep learning approaches for predicting protein-protein interactions within the ATP synthase complex

• Network analysis:

  • Modeling the regulatory networks controlling ATP synthase expression

  • Investigating co-evolution patterns between ATP synthase components

These computational approaches complement experimental studies by providing mechanistic insights at temporal and spatial scales often inaccessible to direct experimental observation .