Recombinant Bacillus cereus ATP synthase subunit alpha (atpA), partial

What is the structure and function of Bacillus cereus ATP synthase subunit alpha (atpA)?

The alpha subunit is one of the five components (α, β, δ, γ, and ε) that form the F1 complex of ATP synthase in B. cereus, which bears the catalytic site for ATP synthesis . In bacterial ATP synthases, this F1 complex connects to the membrane-embedded F0 complex, consisting of subunits a, b, and c . The alpha subunit contributes to the structural stability of the enzyme and participates in the conformational changes necessary for catalysis. While the beta subunits contain the primary catalytic sites, the alpha subunits participate in nucleotide binding and help regulate the enzymatic activity through allosteric mechanisms.

What expression systems are most suitable for producing recombinant B. cereus atpA?

Based on research with bacterial ATP synthase subunits, common expression systems include:

For optimal expression in E. coli systems, induction at lower temperatures (16-20°C) with reduced IPTG concentration (0.1-0.5 mM) often improves solubility of recombinant ATP synthase subunits.

What purification strategy yields the highest purity and activity for recombinant B. cereus atpA?

A multi-step purification approach is recommended:

• Affinity chromatography: If using a His-tagged construct, nickel affinity chromatography provides an effective initial capture step.

• Ion exchange chromatography: Based on the theoretical isoelectric point of atpA, anion exchange at pH 7.5-8.0 can provide further purification.

• Size exclusion chromatography: As a final polishing step to remove aggregates and achieve high homogeneity.

Buffer composition significantly impacts stability and activity. Consider including:

• ATP (1-2 mM) as a stabilizing ligand

• Magnesium chloride (5-10 mM) as a cofactor

• HEPES or Tris buffer (50 mM, pH 7.5-8.0)

• Sodium chloride (100-200 mM) for stability

• Reducing agent (DTT, 1-5 mM) to prevent oxidation

• Glycerol (10-20%) for long-term storage

How can researchers assess the functionality of recombinant B. cereus atpA?

Since the alpha subunit alone does not exhibit catalytic activity, functional assessment typically requires:

• Nucleotide binding assays:

  • Fluorescence-based methods using fluorescent ATP analogs

  • Isothermal titration calorimetry to determine binding constants

  • Surface plasmon resonance for binding kinetics

• Reconstitution studies:

  • Co-expression or reconstitution with other ATP synthase subunits

  • Assessment of ATPase activity in the reconstituted complex

  • Proton pumping assays using reconstituted proteoliposomes

• Structural integrity assessment:

  • Circular dichroism spectroscopy to verify secondary structure

  • Thermal stability assays to evaluate folding quality

  • Limited proteolysis to identify stable domains

It's important to note that recombinant partial atpA may not exhibit the same functional properties as the native subunit within the complete ATP synthase complex.

What role does ATP synthase play in B. cereus acid stress response?

Recent research indicates that ATP synthase activity is crucial for B. cereus acid stress response. When B. cereus cells are exposed to acid stress, ATPase activity leads to an increase in intracellular pH . Expression of ATP synthase genes is increased in acid-adapted cells compared to non-adapted cells before and after acid shock .

The F1F0-ATPase in B. cereus appears to function as a proton pump during acid stress, driving protons out of the cell with parallel hydrolysis of ATP, similar to its role in E. coli . This mechanism allows B. cereus to induce an acid tolerance response (ATR) that depends on ATPase activity induction and intracellular pH homeostasis . This adaptation also provides cross-protection against other stresses such as ethanol stress and heat stress .

What structural differences exist between bacterial and eukaryotic ATP synthase alpha subunits?

The bacterial ATP synthase, including B. cereus atpA, represents a simpler form of the enzyme that performs the same core functions as the more complex mitochondrial counterparts . Key differences include:

• Regulatory mechanisms: The bacterial enzyme has fewer regulatory subunits, with regulation often mediated through the ε subunit in an ATP-dependent manner .

• Inhibitory states: In Bacillus PS3, low ATP concentrations (<0.7 mM) promote an inhibitory "up" conformation of subunit ε, while high ATP concentrations (>1 mM) induce a permissive "down" conformation . This mechanism allows the ATP synthase to run in reverse, establishing a proton motive force by ATP hydrolysis, only when ATP is abundant .

• Structural architecture: In bacterial enzymes, loops in subunit a fill the role of additional subunits found in the F0 region of mitochondrial enzymes .

These differences could potentially be exploited for the development of selective inhibitors targeting bacterial ATP synthases.

How does the mechanism of proton translocation in B. cereus ATP synthase differ from other species?

Recent structural studies of bacterial ATP synthases have revealed the path of transmembrane proton translocation . While specific details for B. cereus are not fully characterized, studies on related bacterial ATP synthases provide insights:

• The proton path likely involves a series of charged and polar residues in the a and c subunits of the F0 complex.

• The rotation of the c-ring is coupled to conformational changes in the F1 complex, including the alpha subunit, which drives ATP synthesis.

• Unlike some bacterial species, B. cereus F1F0-ATPase activity is insensitive to DCCD , suggesting a potentially unique mechanism or structural feature in the proton translocation pathway.

• The enzyme's role in acid tolerance suggests specific adaptations that allow it to function effectively under acidic conditions, potentially involving unique residues in the proton channel.

What are common challenges in expressing and purifying recombinant B. cereus atpA?

Researchers frequently encounter these obstacles:

• Solubility issues:

  • Tendency to form inclusion bodies in E. coli

  • Misfolding due to absence of partner subunits

  • Solution: Lower induction temperature, use solubility tags (SUMO, MBP), co-expression with chaperones

• Stability concerns:

  • Degradation during purification

  • Loss of activity during storage

  • Solution: Include protease inhibitors, optimize buffer components, store with nucleotides and glycerol

• Functional reconstitution:

  • Difficulty assembling with other subunits

  • Challenges in measuring activity of partial protein

  • Solution: Co-expression strategies, careful design of constructs to include interface regions

• Protein heterogeneity:

  • Multiple conformational states

  • Aggregation during concentration

  • Solution: Add stabilizing ligands, optimize buffer conditions, use analytical SEC to monitor homogeneity

How can researchers differentiate between the roles of different ATP synthase subunits in B. cereus?

Distinguishing the specific contributions of atpA from other ATP synthase subunits requires sophisticated approaches:

• Genetic approaches:

  • Site-directed mutagenesis targeting residues unique to atpA

  • Chimeric constructs swapping domains between related species

  • Complementation studies in ATP synthase-deficient strains

• Biochemical methods:

  • Reconstitution experiments with defined subunit composition

  • Crosslinking studies to identify interacting partners

  • Activity assays with specific inhibitors

• Structural studies:

  • Cryo-EM of the intact complex in different conformational states

  • X-ray crystallography of subcomplexes

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• Computational approaches:

  • Molecular dynamics simulations to predict subunit interactions

  • Sequence conservation analysis to identify functionally important residues

  • Homology modeling based on related bacterial ATP synthases

What are the best approaches for studying the role of atpA in B. cereus acid stress response?

Investigating atpA's contribution to acid tolerance requires multiple complementary approaches:

• Gene expression analysis:

  • qRT-PCR to measure atpA expression under acid stress

  • Transcriptomics to identify co-regulated genes

  • Promoter reporter fusions to study regulation

• Protein-level studies:

  • Western blotting to quantify protein levels during acid adaptation

  • Phosphoproteomic analysis to identify potential regulatory modifications

  • In vitro activity assays at different pH values

• Physiological studies:

  • pH homeostasis measurements in wild-type versus atpA mutant strains

  • Survival assays under acid challenge

  • ATP levels and proton motive force determination

• Structure-function analysis:

  • Identification of pH-sensitive residues

  • Mutagenesis of potential proton-sensing sites

  • Comparison with acid-tolerant versus acid-sensitive bacterial species

How might high-resolution structural studies of B. cereus ATP synthase advance antimicrobial development?

ATP synthase represents a potential target for novel antimicrobials, with several promising research directions:

• Structure-based drug design:

  • High-resolution structures of B. cereus ATP synthase could reveal unique pockets for selective inhibitor binding

  • Targeting the interface between atpA and other subunits might disrupt complex assembly

  • Compounds that lock the enzyme in an inhibitory conformation could be effective antimicrobials

• Exploiting species-specific differences:

  • Structural features unique to bacterial enzymes versus human homologs

  • B. cereus-specific regulatory mechanisms

  • Differences in proton translocation pathways

• Combination strategies:

  • ATP synthase inhibitors could potentially sensitize B. cereus to conventional antibiotics

  • Targeting energy production might overcome certain resistance mechanisms

  • Dual targeting of ATP synthesis and acid tolerance pathways

The insensitivity of B. cereus F1F0-ATPase to DCCD , a common inhibitor of ATP synthases, suggests unique structural features that could be exploited for selective targeting.

What emerging technologies are advancing research on bacterial ATP synthase subunits?

Several cutting-edge methodologies are transforming ATP synthase research:

• Cryo-electron microscopy:

  • Recent advances have enabled visualization of bacterial ATP synthases at ~3.0 Å resolution in different rotational states

  • Allows observation of conformational changes without crystallization

  • Can capture multiple functional states of the complex

• Single-molecule techniques:

  • FRET to monitor conformational dynamics

  • Magnetic tweezers to study rotational mechanics

  • Single-molecule force spectroscopy to investigate subunit interactions

• Mass spectrometry methods:

  • Hydrogen-deuterium exchange to probe dynamic regions

  • Crosslinking mass spectrometry to map interaction interfaces

  • Native mass spectrometry for intact complex analysis

• Computational approaches:

  • Molecular dynamics simulations of proton translocation

  • Machine learning for prediction of mutation effects

  • Systems biology modeling of ATP synthase in cellular energy networks

How does recombinant B. cereus atpA research contribute to understanding bacterial adaptation mechanisms?

Research on B. cereus ATP synthase provides broader insights into bacterial adaptation:

• Energy metabolism during stress:

  • ATP synthase activity is critical for acid stress response in B. cereus

  • Understanding how energy production systems adapt to environmental challenges

  • Potential connections between ATP homeostasis and virulence

• Evolution of bacterial energy systems:

  • Comparative studies between B. cereus and related species

  • Identification of conserved versus variable elements in ATP synthase structure

  • Understanding how the "simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex"

• Host-pathogen interactions:

  • Role of ATP synthase in survival within acidic host environments

  • Contribution to persistence during infection

  • Potential as a vaccine target or diagnostic marker

These research directions highlight how fundamental studies of recombinant B. cereus atpA contribute to both basic science understanding and potential applied outcomes in antimicrobial development and biotechnology.