Recombinant Bifidobacterium adolescentis ATP synthase subunit beta (atpD)

What is the biological significance of ATP synthase subunit beta (atpD) in Bifidobacterium adolescentis?

The ATP synthase subunit beta (atpD) in B. adolescentis is a critical component of the F-type ATP synthase complex that catalyzes ATP synthesis through oxidative phosphorylation. This protein plays an essential role in energy metabolism, allowing B. adolescentis to adapt to the environmental conditions of the human gut. Based on evolutionary studies, the F-type ATP synthase was present in the Last Bacterial Common Ancestor (LBCA), establishing its fundamental importance in bacterial metabolism . Unlike some Archaea that possess A/V-type ATP synthases, B. adolescentis as a bacterial species typically contains F-type ATP synthases which are integral to its energy production capabilities and survival in the gastrointestinal tract .

How prevalent is Bifidobacterium adolescentis in the human gut microbiome?

B. adolescentis is one of the most abundant bifidobacterial species in the adult human gut microbiome. Metagenomic analyses of 4,019 human gut samples from healthy adults revealed that B. adolescentis has an average relative abundance of 1.52% ± 0.06% with a prevalence of 42.0% across adult populations . In elderly individuals (over 80 years), the species maintains its significance with an average abundance of 1.64% ± 0.23% and a prevalence of 29.5% . This data positions B. adolescentis alongside B. longum as key bifidobacterial contributors to the human gut microbiota from adolescence through to old age, highlighting its potential importance as a model organism for microbiome research.

The relative abundances of various Bifidobacterium species in human gut microbiomes is summarized in the following table:

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

For recombinant production of B. adolescentis atpD, several expression systems can be considered, with Escherichia coli being the most commonly used for initial attempts due to its well-established protocols and high yields. The optimal expression system selection should be based on factors including protein folding requirements, post-translational modifications, and intended applications of the recombinant protein .

When working with bacterial proteins like atpD, E. coli BL21(DE3) and its derivatives are often the first choice due to their reduced protease activity and the availability of T7 promoter-based expression systems. For proteins requiring specific chaperones or disulfide bond formation, strains like Origami or SHuffle may be more appropriate. If the native protein interacts with other ATP synthase subunits for proper folding, co-expression strategies might be necessary .

Alternative expression systems including Bacillus subtilis or Lactococcus lactis could be considered for proteins that may be toxic in E. coli or require specific processing conditions. These Gram-positive hosts might provide an environment more similar to the native Bifidobacterium conditions .

How can Design of Experiments (DoE) be applied to optimize recombinant B. adolescentis atpD expression?

Design of Experiments (DoE) offers a systematic approach to optimize recombinant B. adolescentis atpD expression by simultaneously evaluating multiple factors affecting protein production. Unlike the one-factor-at-a-time approach, DoE enables researchers to identify not only individual factor effects but also interaction effects between variables with a carefully selected small set of experiments .

For atpD expression optimization, a factorial design or response surface methodology (RSM) could be implemented to investigate critical factors including:

• Induction parameters (inducer concentration, induction timing, temperature)

• Media composition (carbon source, nitrogen content, mineral supplements)

• Environmental conditions (pH, aeration, agitation speed)

• Host strain genetic background

The optimization process would involve:

• Screening phase: Use fractional factorial designs to identify the most influential factors among many potential variables

• Optimization phase: Apply RSM to develop mathematical models describing how the significant factors affect protein yield and quality

• Validation phase: Confirm the model's predictions with experimental verification under the predicted optimal conditions

This approach allows researchers to develop a comprehensive understanding of the expression system dynamics while minimizing experimental costs and time investment. Statistical analysis of the results using specialized software packages enables the identification of optimal conditions for maximum atpD yield and quality .

What are the evolutionary implications of studying ATP synthase subunit beta across different bacterial species?

Studying ATP synthase subunit beta (atpD) across bacterial species, including B. adolescentis, provides valuable insights into evolutionary relationships and functional adaptations. Phylogenetic analyses have confirmed that F-type ATP synthases were present in the Last Bacterial Common Ancestor (LBCA), while A/V-type ATP synthases were present in the Last Archaeal Common Ancestor (LACA) .

Interestingly, evolutionary studies have revealed that A/V-type ATP synthases are broadly distributed in Bacteria and might have already been present in LBCA, challenging previous assumptions about their exclusive archaeal origin . Conversely, only three archaeal genomes (all within the genus Methanosarcina) appear to encode F-type ATP synthases, suggesting rare horizontal gene transfer events in the opposite direction .

For B. adolescentis specifically, comparative analysis of its atpD sequence with other gut microbiome members could reveal:

• Selective pressures specific to the human gut environment

• Potential functional adaptations related to energy metabolism in low-oxygen conditions

• Evidence of horizontal gene transfer events within the gut microbiome

• Molecular evolution rates in conserved protein domains versus variable regions

These evolutionary insights could guide the design of mutagenesis experiments targeting conserved residues to understand structure-function relationships and potentially engineer enzymes with altered properties for biotechnological applications.

How does the ecological context of B. adolescentis influence atpD expression and function?

The ecological context of B. adolescentis as a prominent member of the human gut microbiota has significant implications for atpD expression and function. B. adolescentis has evolved to thrive in the complex and competitive environment of the adult human gut, which influences its energy metabolism strategies and, consequently, ATP synthase activity .

In vitro experiments with the prototype strain B. adolescentis PRL2023 have demonstrated its ability to survive gastrointestinal tract conditions and interact with both intestinal cells and other microbial gut commensals . Co-cultivation studies revealed that B. adolescentis engages in various microbe-microbe interactions and can co-metabolize plant-derived glycans like xylan .

These ecological adaptations likely influence atpD expression through:

• Metabolic flexibility: The ability to switch carbohydrate utilization pathways depending on available substrates may necessitate regulated ATP synthase expression to balance energy needs

• Stress response: Adaptation to gut environmental stressors (pH fluctuations, oxygen gradients, nutrient limitation) may impact ATP synthase regulation

• Competitive pressure: Interactions with other gut microbes may drive selection for efficient energy metabolism systems

Understanding these ecological factors can inform experimental design when studying recombinant atpD, particularly regarding growth conditions that mimic the native environment and regulation of gene expression under different simulated gut conditions.

What strategies can be employed to enhance solubility of recombinant B. adolescentis atpD?

Enhancing the solubility of recombinant B. adolescentis atpD requires a multifaceted approach addressing protein expression, folding, and stabilization. Several strategies can be implemented and optimized using DoE methodologies:

• Expression temperature modulation: Lower temperatures (15-25°C) often improve folding by slowing translation rates, allowing more time for proper protein folding. This can be particularly important for complex proteins like ATP synthase subunits .

• Co-expression with molecular chaperones: Systems expressing folding assistants such as GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor can facilitate proper folding of atpD.

• Fusion tag selection: Solubility-enhancing tags such as MBP (maltose-binding protein), SUMO, or Thioredoxin can significantly improve soluble expression. A systematic comparison of different fusion partners using a DoE approach could identify the optimal tag for atpD.

• Buffer composition optimization: Screening various buffer components, ionic strengths, and additives during purification can stabilize the recombinant protein. Common additives include:

  • Mild detergents (0.05-0.1% Triton X-100)

  • Osmolytes (glycerol, sucrose)

  • Salt concentration variations (100-500 mM NaCl)

  • Divalent cations (particularly important for ATP-binding proteins)

• Codon optimization: Adapting the codon usage of the atpD gene to the expression host can prevent translation stalling and improve expression efficiency.

The optimal combination of these factors can be determined through systematic DoE approaches, testing interactions between variables to identify conditions that maximize soluble protein yield .

How can researchers validate the functional activity of recombinant B. adolescentis atpD?

Validating the functional activity of recombinant B. adolescentis atpD presents unique challenges since the beta subunit is part of the larger ATP synthase complex. Several complementary approaches can be implemented:

• ATP binding assays: As the beta subunit contains the catalytic nucleotide-binding site, radioactive or fluorescent ATP analogs can be used to measure binding affinity. Techniques include:

  • Isothermal titration calorimetry (ITC)

  • Fluorescence anisotropy with labeled ATP

  • Surface plasmon resonance (SPR)

• ATPase activity measurements: While isolated beta subunits typically show reduced activity compared to the complete complex, basal ATPase activity can be assessed using:

  • Coupled enzyme assays (with pyruvate kinase and lactate dehydrogenase)

  • Colorimetric phosphate detection methods

  • Luciferase-based ATP consumption assays

• Structural validation: Proper folding can be confirmed through:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure

  • Intrinsic tryptophan fluorescence to assess tertiary structure

  • Size exclusion chromatography to evaluate oligomeric state

• Complementation studies: Expression of recombinant atpD in ATP synthase-deficient bacterial strains can demonstrate functional activity through growth restoration under conditions requiring oxidative phosphorylation.

• Interaction studies: Assessing the ability of the recombinant atpD to interact with other ATP synthase subunits using:

  • Pull-down assays

  • Co-immunoprecipitation

  • Yeast two-hybrid or bacterial two-hybrid systems

A comprehensive validation approach would include multiple methods to confirm both structural integrity and functional competence of the recombinant protein.

How can Google's People Also Ask (PAA) data inform research questions about B. adolescentis atpD?

Google's People Also Ask (PAA) feature provides valuable insights into the questions researchers and the scientific community are asking about specific topics. For B. adolescentis atpD research, PAA data can inform experimental design and identify knowledge gaps through the following approach:

• Systematic query analysis: Researchers can use PAA data to identify recurring themes and questions surrounding ATP synthase in Bifidobacterium and related organisms. This reveals the current focus areas and potential blind spots in the field .

• Intent proximity mapping: PAA reveals questions that are closely related by "intent proximity," meaning they frequently occur together when researchers are investigating a topic. For atpD research, this could highlight connections between protein structure, function, expression systems, and ecological relevance that might not be immediately apparent .

• Knowledge gap identification: Questions that appear in PAA but lack high-quality answer snippets may indicate areas where research is needed. By analyzing these patterns, researchers can identify understudied aspects of B. adolescentis atpD .

• Research trend monitoring: Regular monitoring of PAA data over time reveals how research interests evolve, helping scientists stay at the forefront of emerging questions. This is particularly valuable for tracking changes in research focus following new discoveries or technological advances .

To implement this approach, researchers should:

• Perform broad queries about "Bifidobacterium adolescentis ATP synthase" and related terms

• Document all PAA questions that appear

• Categorize questions by research domain (structural, functional, ecological, methodological)

• Identify clusters of related questions that may inform comprehensive research approaches

• Revisit PAA data periodically to track changes in research interests

What statistical approaches are most appropriate for analyzing optimization experiments with recombinant B. adolescentis atpD?

When analyzing optimization experiments for recombinant B. adolescentis atpD expression, several statistical approaches are particularly effective depending on the experimental design implemented:

• For factorial designs:

  • Analysis of Variance (ANOVA) to determine which factors and interactions significantly affect protein yield

  • Main effects and interaction plots to visualize the impact of different factors

  • Pareto charts of standardized effects to rank factors by importance

• For response surface methodology (RSM):

  • Regression analysis to develop predictive models (typically second-order polynomial equations)

  • Response surface plots and contour plots to visualize optimal conditions

  • Canonical analysis to characterize the response surface (maximum, minimum, or saddle point)

• For all experimental designs:

  • Residual analysis to validate model assumptions (normality, homoscedasticity)

  • Lack-of-fit tests to assess model adequacy

  • Prediction intervals to estimate the uncertainty in future observations

The analysis process should follow these steps:

• Data transformation if necessary (log, square root) to meet statistical assumptions

• Model fitting using appropriate software (e.g., Design-Expert, JMP, R with specialized packages)

• Model reduction by removing non-significant terms

• Diagnostic checking to validate the final model

• Numerical optimization to identify conditions that maximize protein yield and/or quality metrics

How might strain differences in B. adolescentis affect recombinant atpD production and function?

Key considerations regarding strain differences include:

• Genetic variability: Comparative genomic analyses have revealed variations in gene content and organization even within the same bifidobacterial species. These differences may affect:

  • Promoter sequences influencing native atpD expression

  • Codon usage patterns affecting translation efficiency

  • Post-translational modification capabilities

  • Protein folding machinery and chaperone systems

• Physiological adaptations: Different B. adolescentis strains show varying abilities to:

  • Survive gastrointestinal conditions (pH tolerance, bile resistance)

  • Metabolize specific carbohydrates (affecting energy requirements)

  • Interact with host cells and other microbiota members

  • Respond to environmental stressors

• Implications for recombinant production: When designing expression systems for atpD, researchers should:

  • Consider the codon optimization strategy based on the specific source strain

  • Evaluate if strain-specific post-translational modifications are necessary for function

  • Assess if co-factors or interacting proteins differ between strains

• Functional validation: When characterizing recombinant atpD, strain-specific considerations include:

  • Comparing enzymatic parameters (Km, Vmax) between atpD variants

  • Assessing structural differences through biophysical techniques

  • Evaluating interaction capabilities with other ATP synthase components

Understanding these strain-specific nuances is essential for accurate interpretation of research findings and development of optimized expression systems .

What potential applications exist for engineered variants of B. adolescentis atpD?

Engineered variants of B. adolescentis atpD could serve multiple research and biotechnological purposes:

• Structure-function relationship studies:

  • Site-directed mutagenesis of catalytic residues to elucidate reaction mechanisms

  • Domain swapping with ATP synthases from other organisms to identify determinants of substrate specificity

  • Introduction of fluorescent probes at strategic positions to monitor conformational changes during catalysis

• Bioenergetic research tools:

  • Creation of atpD variants with altered nucleotide specificity for studying energy transfer in cells

  • Development of inhibitor-resistant variants to probe regulation of oxidative phosphorylation

  • Engineering variants with enhanced stability for structural studies of the ATP synthase complex

• Biotechnological applications:

  • Design of atpD variants with improved catalytic efficiency for ATP production in cell-free systems

  • Development of sensors for ATP detection based on engineered atpD binding domains

  • Creation of thermal-stable variants for industrial applications requiring robust ATP regeneration

• Probiotics development:

  • Engineering B. adolescentis strains with optimized atpD to enhance survival in the gastrointestinal tract

  • Modifying energy metabolism through atpD engineering to improve competitive fitness in probiotic formulations

  • Creating reporter strains with tagged atpD to monitor probiotic localization and activity in vivo

These applications leverage the fundamental role of atpD in energy metabolism while exploiting the ecological significance of B. adolescentis in the human gut microbiome .