Recombinant Bifidobacterium adolescentis ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Bifidobacterium adolescentis?

Subunit c (atpE) in B. adolescentis is a highly conserved membrane protein that forms the c-ring of the F₀ domain in the F₁F₀-ATP synthase complex. This ring is embedded in the cellular membrane and functions as the ion-translocating component. Like other bacterial ATP synthases, the B. adolescentis enzyme likely consists of two main subcomplexes - the membrane-extrinsic F₁ region (containing α₃β₃γδε subunits) and the membrane-intrinsic F₀ region (containing subunits a, b, and the c-ring) .

The c-ring rotation, driven by proton translocation across the membrane, is mechanically coupled to ATP synthesis in the F₁ domain. In many bifidobacteria that lack a respiratory chain, the primary role of the F₁F₀-ATPase is typically to create a proton gradient through ATP hydrolysis rather than ATP synthesis . This physiological function is particularly important for maintaining pH homeostasis in the acidic intestinal environment where B. adolescentis naturally resides.

Why is the atp operon significant for Bifidobacterium research?

The atp operon has emerged as an important molecular marker for bacterial classification and phylogenetic analysis, complementing the widely used 16S rRNA gene approach . In bifidobacteria, the atp operon (typically arranged as atpBEFHAGDC) is highly conserved, making it valuable for species identification and evolutionary studies .

For researchers studying B. adolescentis, the atp operon provides:

• Alternative taxonomic markers with higher resolution than 16S rRNA for distinguishing closely related strains

• Insights into the adaptation of bifidobacteria to acidic environments

• Understanding of energy metabolism in these beneficial gut microbes

• Potential targets for genetic manipulation to enhance probiotic properties

Sequence analysis of the atpD gene (encoding the β subunit) has been used successfully for classification of bifidobacterial species, indicating that other atp genes including atpE could serve similar taxonomic purposes .

How does the c-ring stoichiometry affect ATP synthase function in bifidobacteria?

The c-ring stoichiometry (number of c subunits per ring) directly determines the ion-to-ATP ratio and significantly impacts the bioenergetic efficiency of the ATP synthase. While the exact c-ring stoichiometry in B. adolescentis has not been definitively determined, bacterial c-rings typically contain between 10-15 subunits .

This stoichiometry establishes the coupling ratio between ion translocation and ATP synthesis/hydrolysis. For each complete rotation of the c-ring, three ATP molecules are synthesized or hydrolyzed (due to the three catalytic sites in the F₁ α₃β₃ hexamer). Therefore, if B. adolescentis has a c₁₀ ring, the coupling ratio would be 10H⁺:3ATP (approximately 3.3:1). If it has a c₁₅ ring, the ratio would be 15H⁺:3ATP (5:1) .

The stoichiometric variation between species is thought to reflect adaptation to different energetic demands and environmental conditions. Organisms adapted to energy-limited environments might evolve larger c-rings to maximize the energy extracted from small ion gradients, while those in energy-rich environments might prioritize rapid ATP synthesis with smaller rings.

What expression systems are suitable for producing recombinant B. adolescentis atpE?

Several expression systems can be considered for recombinant production of B. adolescentis atpE, with E. coli being the most widely used heterologous host. Based on challenges encountered with other c subunits, researchers should consider these approaches:

1. Fusion protein expression:
The hydrophobic nature of subunit c often creates expression and solubility challenges. Expression as a fusion with maltose-binding protein (MBP) has proven successful for other c subunits, as seen with the chloroplast ATP synthase c subunit that could only be expressed when fused to MBP . The vector pMAL-c2x carrying the malE gene fused to atpE provides a viable expression system .

2. Homologous expression:
Expression within Bifidobacterium itself could preserve native folding and post-translational modifications. Techniques developed for other Bifidobacterium species, such as the system used for BopA protein expression in B. bifidum S17 , could potentially be adapted for B. adolescentis atpE.

3. Cell-free protein synthesis:
For difficult membrane proteins like atpE, cell-free systems incorporating lipid nanodiscs or detergent micelles might facilitate proper folding while avoiding toxicity issues associated with membrane protein overexpression.

Selection of the appropriate system should be guided by the research objectives, required protein yield, and whether functional studies will be performed with the recombinant protein.

How can recombinant atpE be used to investigate pH adaptation mechanisms in B. adolescentis?

The F₁F₀-ATPase in lactic acid bacteria and bifidobacteria plays a crucial role in acid stress response, with increased activity at lower environmental pH. Recombinant atpE provides a powerful tool to study this adaptation mechanism through several experimental approaches:

Site-directed mutagenesis studies:
Researchers can identify key residues involved in proton binding and translocation by introducing specific mutations into recombinant atpE. By expressing these mutants in either E. coli or B. adolescentis and measuring ATPase activity under varying pH conditions, the molecular basis of pH sensitivity can be elucidated.

c-ring reconstitution experiments:
Purified recombinant atpE can be used to reconstitute c-rings in vitro. These reconstructed rings can then be incorporated into liposomes to measure proton translocation at different pH values. Comparison with c-rings from acid-tolerant versus acid-sensitive bacterial species can reveal structural adaptations specific to B. adolescentis.

Transcriptional regulation analysis:
The regulation of the atp operon in response to acidic conditions appears to occur at the transcriptional level in some bifidobacteria . Using recombinant reporter systems fused to the atp operon promoter, researchers can identify regulatory elements that control pH-dependent expression of atpE and other ATP synthase components.

These approaches can provide insights into how B. adolescentis maintains energy homeostasis in the acidic gut environment, which has implications for its probiotic applications and survival during gastrointestinal transit.

What methodological challenges exist in determining the c-ring stoichiometry of B. adolescentis ATP synthase?

Determining the exact c-ring stoichiometry in B. adolescentis ATP synthase presents several technical challenges that researchers must address:

Challenge 1: Protein isolation
The membrane-embedded nature of the c-ring makes isolation difficult without disrupting its native structure. Additionally, the hydrophobicity of subunit c creates solubility issues during purification.

Methodological solution:

• Use gentle detergent solubilization (e.g., n-dodecyl-β-maltoside) followed by blue native PAGE to preserve the intact c-ring

• Apply a recombinant approach with cysteine cross-linking, where strategically placed cysteine residues can lock the c-ring structure during solubilization

• Consider on-column refolding techniques for recombinant atpE to promote proper oligomerization

Challenge 2: Structural analysis
The small size and hydrophobic nature of the c-ring make high-resolution structural determination challenging.

Methodological solution:

• Employ atomic force microscopy (AFM) of reconstituted c-rings in lipid bilayers

• Use negative-stain electron microscopy followed by single-particle analysis for initial structural characterization

• For definitive stoichiometry, apply mass determination methods such as mass photometry or native mass spectrometry

Challenge 3: Functional verification
Confirming that the observed stoichiometry represents the physiologically relevant form.

Methodological solution:

• Develop reconstituted proteoliposome systems with purified components to measure ion translocation and ATP synthesis/hydrolysis ratios

• Apply metabolic control analysis to correlate c-ring stoichiometry with bioenergetic parameters in living cells

• Use genetic approaches to lock the c-ring at defined stoichiometries and measure functional consequences

How can evolutionary analysis of atpE sequences inform our understanding of bioenergetic adaptation in Bifidobacterium species?

Evolutionary analysis of atpE sequences across Bifidobacterium species can reveal important insights into bioenergetic adaptation. A comprehensive approach should include:

Phylogenetic analysis:
By constructing phylogenetic trees based on atpE sequences from multiple bifidobacterial species and strains, researchers can identify evolutionary relationships and potential adaptation events. This approach has been successfully applied to atpD sequences in bifidobacteria, establishing specific sequence signatures for the genus Bifidobacterium and closely related taxa .

Selection pressure analysis:
Calculation of dN/dS ratios (non-synonymous to synonymous substitution rates) across the atpE gene can identify regions under positive selection, indicating adaptive evolution. Particular focus should be placed on residues involved in ion binding and c-ring assembly.

Structural homology modeling:
Using resolved structures from other bacterial c-rings as templates, homology models of B. adolescentis atpE can be constructed to map conserved and variable regions onto the protein structure. This approach can identify structurally significant variations between species adapted to different environmental niches.

Correlation with ecological parameters:
Statistical analysis correlating sequence variations with ecological parameters (pH tolerance, preferred carbon sources, host specificity) can reveal environment-specific adaptations. For example, bifidobacterial species found in the infant gut versus those in the adult gut might show distinct evolutionary patterns in their atpE sequences reflecting adaptation to different intestinal conditions.

Note: This table contains predicted characteristics based on existing knowledge of bacterial ATP synthases; the exact stoichiometry values for Bifidobacterium species require experimental verification.

What are the critical parameters for optimizing functional reconstitution of recombinant B. adolescentis atpE into liposomes?

Functional reconstitution of recombinant B. adolescentis atpE into liposomes is essential for biophysical and enzymatic studies. The following parameters require careful optimization:

Lipid composition:
The membrane environment significantly affects c-ring assembly and function. Researchers should test:

• Different phospholipid compositions (varying ratios of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol)

• Incorporation of bacterial lipids, particularly those found in Bifidobacterium membranes

• Lipid bilayer thickness, which must match the hydrophobic length of the c-ring

Reconstitution method:
Several approaches can be considered:

• Detergent-mediated reconstitution followed by detergent removal via dialysis or Bio-Beads

• Direct incorporation during liposome formation

• Fusion of proteoliposomes with preformed liposomes

Buffer conditions:
Critical buffer parameters include:

• pH (testing physiologically relevant range: 5.0-7.5)

• Ionic strength (50-200 mM KCl or NaCl)

• Presence of stabilizing agents (glycerol, sucrose)

• Divalent cations (Mg²⁺, Ca²⁺) at varying concentrations

Protein-to-lipid ratio:
The optimal protein-to-lipid ratio must be determined empirically, typically testing a range from 1:50 to 1:2000 (w/w). Too high a protein concentration may lead to aggregation, while too low a concentration may result in insufficient signal in functional assays.

Functional verification:
Successful reconstitution should be verified by:

• Freeze-fracture electron microscopy to visualize incorporated protein

• Proton/sodium ion transport assays using pH-sensitive or Na⁺-sensitive fluorescent dyes

• ATP synthesis/hydrolysis measurements under appropriate ion gradient conditions

Similar reconstitution approaches have been successfully applied to the Na⁺ A₁A₀ ATP synthase from Eubacterium limosum, enabling demonstration of ATP-dependent Na⁺ transport and Na⁺-driven ATP synthesis after incorporation into liposomes .

What purification strategy is most effective for recombinant B. adolescentis atpE?

A multi-step purification strategy is recommended for obtaining pure, functional recombinant B. adolescentis atpE:

Step 1: Expression optimization
Based on experiences with other c subunits, expression as an MBP fusion protein is likely to be most successful . The optimal conditions include:

• IPTG concentration: 0.1-0.5 mM

• Induction temperature: 18-25°C (lower temperatures to reduce inclusion body formation)

• Induction time: 16-20 hours

• Host strain: C41(DE3) or C43(DE3), specifically developed for membrane protein expression

Step 2: Initial extraction and clarification

• Cell disruption via sonication or high-pressure homogenization in buffer containing detergent (e.g., n-dodecyl-β-maltoside at 1%)

• Centrifugation at 20,000×g to remove cell debris

• Ultracentrifugation at 100,000×g to collect membrane fraction (if using membrane-bound expression strategy)

Step 3: Affinity chromatography
For His-tagged constructs:

• Ni-NTA affinity chromatography with gradual imidazole elution (20-300 mM)

• Buffer containing appropriate detergent (0.05-0.1% n-dodecyl-β-maltoside)

• Addition of glycerol (10%) to maintain stability

For MBP fusion constructs:

• Amylose resin affinity chromatography

• Elution with maltose (10 mM)

• Optional: TEV protease cleavage to remove MBP tag

Step 4: Size exclusion chromatography

• Superdex 75 or 200 column in buffer containing detergent below critical micelle concentration

• Analysis of oligomeric state and uniformity

Step 5: Ion exchange chromatography (optional)

• For removal of remaining contaminants

• Resource Q or S column depending on theoretical pI of atpE

Quality control:

• SDS-PAGE with silver staining (due to poor Coomassie staining of hydrophobic proteins)

• Mass spectrometry confirmation

• Circular dichroism to verify secondary structure (expected high α-helical content)

• Dynamic light scattering to assess monodispersity

This strategy incorporates approaches successfully used for other ATP synthase c subunits, including the chloroplast c subunit expressed as an MBP fusion protein .

How can researchers assess the functional activity of recombinant B. adolescentis atpE?

Functional assessment of recombinant atpE requires multiple complementary approaches:

1. ATP hydrolysis inhibition assay
Purified recombinant atpE can be tested for its ability to inhibit ATP hydrolysis activity of the F₁ portion of ATP synthase:

• Isolate F₁ portion from B. adolescentis or a related bacterial species

• Measure baseline ATP hydrolysis using colorimetric phosphate release assays

• Add increasing concentrations of purified atpE and measure changes in ATP hydrolysis rate

• A similar approach was used with mycobacterial F₁-ATPase to demonstrate the inhibitory role of subunit ε

2. Proton translocation assays
To assess ion-translocating function after reconstitution into liposomes:

• Load liposomes with pH-sensitive fluorescent dyes (ACMA or pyranine)

• Establish a pH gradient across the membrane

• Monitor fluorescence changes as a measure of proton translocation

• Compare wild-type atpE with site-directed mutants to identify key functional residues

3. DCCD binding studies
N,N'-dicyclohexylcarbodiimide (DCCD) specifically binds to the ion-binding glutamate/aspartate in subunit c:

• Incubate purified atpE with increasing concentrations of DCCD

• Measure binding using fluorescent DCCD derivatives (e.g., NCD-4)

• Determine binding kinetics and competition with protons/sodium ions

• Similar experiments with Na⁺ A₁A₀ ATP synthase demonstrated competition between Na⁺ and DCCD/NCD-4 for a common binding site

4. Reconstitution into complete ATP synthase
The ultimate functional test involves:

• Reconstitution of purified atpE with other ATP synthase subunits

• Measurement of ATP synthesis activity in proteoliposomes under an appropriate ion gradient

• Comparison with native enzyme activity

Structural analysis of c-ring formation

• Blue native PAGE to assess oligomeric assembly

• Electron microscopy of negatively stained or vitrified samples

• Cross-linking studies to analyze subunit interfaces

These methodologies provide complementary data on the structural integrity and functional capacity of recombinant atpE, particularly important for validating site-directed mutants designed to probe structure-function relationships.

What approaches can be used to study the interaction between atpE and other ATP synthase subunits?

Understanding interactions between atpE and other ATP synthase subunits is critical for elucidating the complex's assembly and function. Several complementary approaches can be employed:

Yeast two-hybrid analysis:

• Construct fusion proteins of atpE and other ATP synthase subunits with DNA-binding and activation domains

• Test specific binary interactions in a systematic manner

• Membrane-based yeast two-hybrid systems are preferable for membrane proteins like atpE

Co-immunoprecipitation studies:

• Generate antibodies against atpE or use epitope-tagged versions

• Solubilize ATP synthase complexes under mild conditions

• Immunoprecipitate atpE and identify co-precipitating subunits by western blotting or mass spectrometry

• This approach can identify native interaction partners in B. adolescentis

Cross-linking mass spectrometry:

• Apply chemical cross-linkers of defined lengths to purified ATP synthase or membrane preparations

• Digest cross-linked complexes and analyze by mass spectrometry

• Identify cross-linked peptides to map interaction interfaces

• This method can provide distance constraints for structural modeling

FRET analysis:

• Generate fluorescently labeled versions of atpE and potential interaction partners

• Measure Förster resonance energy transfer in reconstituted systems

• This approach can detect interactions and conformational changes in real-time

Surface plasmon resonance:

• Immobilize purified atpE or potential binding partners on sensor chips

• Measure binding kinetics and affinities in real-time

• Particularly useful for transient interactions during ATP synthase assembly

Genetic approaches:

• Construct chimeric proteins between atpE from different species

• Identify regions necessary for species-specific assembly

• Suppressor mutation analysis to identify compensatory mutations in interacting subunits

The critical interaction between atpE and subunit a deserves particular attention, as this interface forms the pathway for ion translocation. Additionally, interactions with regulatory subunits like ε should be investigated, as this subunit has been implicated in ATPase activity regulation in mycobacterial ATP synthase .

How might genetic manipulation of atpE impact the probiotic properties of B. adolescentis?

Genetic manipulation of atpE could significantly impact several probiotic properties of B. adolescentis through alterations in energy metabolism, acid tolerance, and cellular physiology:

Enhanced acid tolerance:
Strategic modifications to atpE could potentially improve the acid tolerance of B. adolescentis by:

• Optimizing proton pumping efficiency at low pH

• Adjusting the pH response threshold of the ATP synthase

• Enhanced acid tolerance would improve survival during gastrointestinal transit and establishment in the intestinal environment

Altered growth characteristics:
Modifications affecting ATP synthase efficiency could:

• Change growth rates in different environmental conditions

• Alter competitive fitness in the gut ecosystem

• Potentially enhance persistence in the intestinal tract

Metabolic shifts:
Changes in ATP synthase efficiency might redirect carbon flux through:

• Altered fermentation patterns

• Different end-product profiles, potentially affecting host-microbe interactions

• Modified cross-feeding relationships with other microbiota members, similar to how B. adolescentis P2P3 can feed other gut bacteria using resistant starch as a prebiotic

Immunomodulatory effects:
Energy metabolism alterations could impact:

• Production of immunomodulatory factors

• Cell surface properties affecting interaction with host immune cells

• Some B. adolescentis strains have demonstrated the ability to stimulate Th1-type cytokine secretion from macrophages , and these properties might be influenced by metabolic changes

Experimental approaches to explore these effects:

• Site-directed mutagenesis of key residues in atpE

• Heterologous expression of atpE variants from acid-tolerant species

• Controlled expression levels through promoter engineering

• Whole-genome approaches correlating atpE sequence variants with probiotic phenotypes

What insights can comparative analysis of atpE provide about the bioenergetic strategies of different Bifidobacterium species?

Comparative analysis of atpE across Bifidobacterium species can reveal important insights about diverse bioenergetic strategies evolved in different ecological niches:

Habitat-specific adaptations:
Different Bifidobacterium species inhabit distinct niches with varying energy availability and constraints:

• Infant gut specialists (B. infantis, B. breve): Potentially optimized for milk oligosaccharide metabolism

• Adult gut residents (B. adolescentis): Adapted to diverse plant polysaccharides

• Dairy/food isolates (B. animalis): Specialized for growth in food matrices

Comparative atpE analysis can reveal signature adaptations in the ion-translocation machinery that reflect these different energy environments.

Correlation with metabolic capabilities:
Sequence variations in atpE can be correlated with:

• Fermentation patterns (hexose vs. pentose utilization pathways)

• Genome-encoded carbohydrate-active enzymes

• Presence/absence of specific metabolic pathways

Proton vs. sodium coupling:
While most Bifidobacterium species likely use proton-coupled ATP synthases, some bacteria utilize sodium ions instead. Comparative analysis of ion-binding residues in atpE can identify:

• Species potentially utilizing Na⁺ coupling (similar to Eubacterium limosum )

• Evolutionary transitions between H⁺ and Na⁺ specificity

• Bioenergetic implications of ion coupling preference

Structural adaptations:
The c-ring structure, determined by atpE sequences, affects the ion:ATP stoichiometry:

• Species with larger c-rings (more subunits) extract more energy from small ion gradients

• Species with smaller c-rings prioritize rapid ATP synthesis

• These structural adaptations reflect evolutionary optimization for different energetic constraints

Methodological approach for comparative analysis:

• Collect atpE sequences from multiple Bifidobacterium species and strains

• Perform multiple sequence alignment and identification of conserved/variable regions

• Construct phylogenetic trees to identify evolutionary relationships

• Map sequence variations onto structural models to identify functionally significant differences

• Correlate variations with ecological data and phenotypic characteristics

This comprehensive comparative approach could provide a framework for understanding how ATP synthase has evolved within the Bifidobacterium genus to support diverse bioenergetic strategies in different ecological niches.