Recombinant Lactobacillus johnsonii ATP synthase subunit beta (atpD)

What is the structure and function of ATP synthase in Lactobacillus johnsonii?

ATP synthase in L. johnsonii is a multi-subunit protein complex that uses electrochemical proton motive force across the cell membrane to produce ATP from ADP and inorganic phosphate. The enzyme consists of two major domains: the membrane-embedded F₀ domain responsible for proton translocation and the cytoplasmic F₁ domain that catalyzes ATP synthesis .

The ATP synthase complex in bacteria typically follows the gene order atpBEFHAGDC, where atpD encodes the beta subunit of the F₁ domain . This beta subunit contains nucleotide-binding sites and plays a critical role in the catalytic mechanism of ATP synthesis.

ATP synthase in L. johnsonii functions similarly to other bacterial ATP synthases but with specific adaptations for the microaerophilic or anaerobic gut environment. It provides the energy required for various cellular processes, including acid tolerance mechanisms crucial for survival in acidic environments like the gastrointestinal tract .

How is the atpD gene organized within the ATP synthase operon of L. johnsonii?

The atpD gene in L. johnsonii is part of the atp operon that follows the gene order atpBEFHAGDC, which is consistent with most bacterial ATP synthase operons . The complete organization is:

Transcriptional analysis has shown that the atp operon in related bacteria can be transcribed as two separate mRNAs: a complete transcript covering all subunits (approximately 7.3 kb) and a shorter transcript corresponding to the last four genes (atpAGDC) of the operon (approximately 4.5 kb) .

Why is atpD used as a molecular marker for bacterial identification?

The atpD gene has emerged as an effective molecular marker for several key reasons:

• Evolutionary Conservation: The atpD gene is highly conserved among eubacteria due to its essential function, making it suitable for studying evolutionary relationships.

• Sequence Variation: Despite its conservation, atpD contains sufficient sequence variation between species to allow discrimination between closely related bacteria, offering better resolution than 16S rRNA in some cases .

• Signature Sequences: Comparative analysis of atpD nucleotide sequences has revealed species-specific amino acid signatures. For example, specific amino acid signatures have been identified in L. johnsonii and L. gasseri strains that are not found in other microorganisms, enabling species-specific identification .

• Horizontal Gene Transfer Detection: Phylogenetic analysis of atpD genes has shown that the Lactobacillus atpD gene clusters with genera like Listeria, Lactococcus, Streptococcus, and Enterococcus. Its higher G+C content and biased codon usage suggest horizontal gene transfer events, providing insights into bacterial evolution .

For practical applications, atpD-based identification can be implemented through PCR amplification with species-specific primers or restriction fragment length polymorphism (RFLP) analysis targeting the signature sequences .

What are the optimal expression systems for producing recombinant L. johnsonii atpD protein?

The optimal expression system for recombinant L. johnsonii atpD protein is typically Escherichia coli due to its high yield, ease of genetic manipulation, and well-established protocols. Based on available research and commercial products, the following methodological approach is recommended:

• Expression Vector Selection:

  • pET expression systems with N-terminal His-tags have proven effective for ATP synthase subunit expression .

  • The complete coding sequence of L. johnsonii atpD (1-478 amino acids) should be cloned into the expression vector.

• E. coli Strain Selection:

  • BL21(DE3) or its derivatives are preferred for expression of membrane-associated proteins.

  • Rosetta strains may improve expression if rare codons are present in the L. johnsonii sequence.

• Expression Conditions:

  • Induction with IPTG at low concentrations (0.1-0.5 mM) when cultures reach OD₆₀₀ of 0.6-0.8.

  • Expression at lower temperatures (16-25°C) for 12-18 hours improves proper folding.

  • Supplementation with glucose (0.5-1%) can enhance expression yields.

For alternative expression systems, heterologous expression in Lactococcus lactis has been used successfully for other Lactobacillus proteins when authentic post-translational modifications are required .

What purification strategies yield the highest purity and activity of recombinant atpD protein?

A multi-step purification strategy is recommended to obtain high-purity, active recombinant L. johnsonii atpD protein:

• Initial Extraction:

  • For His-tagged constructs, cell lysis using sonication or French press in Tris/PBS-based buffer (pH 8.0) containing 6% Trehalose as used for commercial preparations .

  • Include protease inhibitors and DNase I to prevent degradation and reduce viscosity.

• Affinity Chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins.

  • Gradual imidazole gradient (20-250 mM) for elution to separate weakly bound contaminants.

• Secondary Purification:

  • Ion exchange chromatography (typically Q-Sepharose) to remove remaining impurities.

  • Size exclusion chromatography to isolate monomeric protein and remove aggregates.

• Quality Assessment:

  • SDS-PAGE should confirm >90% purity .

  • Western blotting using anti-His antibodies or specific anti-atpD antibodies.

  • Circular dichroism to verify proper folding.

• Storage:

  • Lyophilized powder form for long-term storage.

  • Reconstituted protein should be supplemented with 5-50% glycerol and stored at -20°C/-80°C in small aliquots to avoid repeated freeze-thaw cycles .

How can recombinant atpD be used to study acid tolerance mechanisms in Lactobacillus species?

Recombinant atpD can be employed to investigate acid tolerance mechanisms in Lactobacillus through several methodological approaches:

• Expression Level Analysis:

  • Northern blot or qRT-PCR analysis of atpD transcripts under different pH conditions.

  • Studies have shown that the atp operon is acid-inducible, with rapid increases in transcript levels upon exposure to low pH, suggesting regulation at the transcriptional level rather than at the enzyme assembly step .

• Functional Complementation Assays:

  • Complement atpD-deficient strains with the recombinant protein to assess restoration of acid tolerance.

  • Compare wild-type and complemented strains' growth rates and survival under acidic conditions.

• Biochemical Activity Assays:

  • ATPase activity can be measured using colorimetric assays that detect inorganic phosphate release.

  • Compare activity at different pH values (ranging from pH 3.5 to 7.0) to determine optimal conditions and pH-dependent regulation.

• Protein-Protein Interaction Studies:

  • Pull-down assays using His-tagged recombinant atpD to identify interaction partners under different pH conditions.

  • This can reveal pH-dependent regulatory mechanisms affecting ATP synthase assembly or activity.

• Site-Directed Mutagenesis:

  • Introduce mutations in conserved residues or species-specific amino acid signatures to study their role in acid adaptation.

  • Amino acid signatures specific to L. johnsonii may be particularly relevant for its unique acid tolerance properties .

Research has shown that ATP synthase plays a crucial role in acid tolerance by extruding protons from the cytoplasm, maintaining intracellular pH homeostasis. Multiple acid tolerance systems have been identified in lactobacilli, including the arginine deiminase (ADI) pathway and amino acid decarboxylation-antiporter reactions, which can generate ATP and enable the extrusion of cytoplasmic protons by the F₀F₁ATPase .

What methodological approaches are used to study the phylogenetic relationships based on atpD sequences?

The atpD gene has proven valuable for phylogenetic analysis of lactic acid bacteria. The following methodological approach is recommended:

• Sequence Amplification and Analysis:

  • PCR amplification using degenerate primers targeting conserved regions of the atpD gene.

  • Full-length sequencing (approximately 1,400 bp) to capture both conserved and variable regions.

• Multiple Sequence Alignment:

  • MUSCLE or CLUSTALW algorithms are suitable for aligning atpD sequences.

  • Manual refinement of alignments may be necessary to account for insertions/deletions.

• Phylogenetic Tree Construction:

  • Maximum Likelihood method with appropriate substitution models (GTR+I+G has been used successfully).

  • Bayesian Inference for more robust statistical support.

  • Include appropriate outgroups and reference sequences from diverse bacterial taxa.

• Signature Sequence Identification:

  • Identify species-specific amino acid signatures through comparative analysis of aligned sequences.

  • For L. johnsonii, eight unique amino acid signatures have been identified that distinguish it from other related species .

• Codon Usage Analysis:

  • Analyze codon usage bias to detect potential horizontal gene transfer events.

  • The Lactobacillus atpD gene shows higher G+C content and biased codon usage compared to the genome average, suggesting horizontal gene transfer from other bacterial lineages .

Phylogenetic analysis based on atpD has successfully distinguished closely related species that are difficult to differentiate using 16S rRNA analysis alone. For example, L. johnsonii and L. gasseri (members of the L. acidophilus B group) branch separately in atpD sequence-based trees .

How does the atpD gene contribute to the energetics and metabolism of L. johnsonii?

The atpD gene, encoding the beta subunit of ATP synthase, plays a central role in the bioenergetics of L. johnsonii, which has specific metabolic adaptations as an obligate homofermentative organism:

• Energy Production in Anaerobic Environments:

  • L. johnsonii is an obligate homofermentative organism that can only undergo homolactic fermentation, converting glucose primarily to lactic acid .

  • Unlike facultative heterofermentative lactobacilli (e.g., L. plantarum), L. johnsonii lacks the pyruvate dehydrogenase complex and other enzymes necessary to convert pyruvate to acetaldehyde, acetyl-CoA, and acetate .

  • ATP synthase provides essential energy through substrate-level phosphorylation and proton motive force utilization.

• ATP Generation Mechanisms:

  • In lactobacilli, ATP can be generated through several pathways:

    • Glycolysis (from glyceraldehyde-3P to pyruvate)

    • Amino acid decarboxylation-antiporter reactions

    • Arginine deiminase (ADI) pathway

• Acid Tolerance and pH Homeostasis:

  • ATP synthase functions in reverse to extrude protons from the cytoplasm, maintaining internal pH.

  • This mechanism requires ATP hydrolysis, highlighting the critical balance between energy production and pH homeostasis.

• Growth Phase-Dependent Regulation:

  • Studies have confirmed that stationary-phase cells are generally more tolerant to low pH than log-phase cells .

  • This suggests phase-dependent regulation of ATP synthase and other acid tolerance mechanisms.

• Metabolic Adaptation:

  • The lack of certain metabolic pathways in L. johnsonii is compensated by enhanced transport systems .

  • ATP synthase activity is therefore critical for energizing various transport processes that acquire nutrients from the environment.

Research has shown that some strains lacking specific components of the AckA-Pta pathway (e.g., phosphate acetyltransferase) can still thrive as strictly homofermentative anaerobic bacteria that undergo homolactic fermentation without requiring acetate for metabolism and ATP production .

What are the structural and functional differences between L. johnsonii atpD and its homologs in other bacterial species?

Comparative analysis reveals several key structural and functional differences between L. johnsonii atpD and its homologs:

• Sequence Conservation and Divergence:

  • Protein comparison shows that ATP synthase subunits from L. johnsonii share greater homology with those from closely related lactobacilli but have significant divergence from more distant taxa like E. coli .

  • Greater homology is observed for the atpA and atpD gene products corresponding to the cytoplasmic domain across species .

  • The lack of amino acid signatures specific to the genus Lactobacillus (in contrast to Bifidobacterium, which has 16 conserved genus-specific signatures) suggests greater variability among Lactobacillus species .

• Structural Differences:

  • The three catalytic β subunits in Bacillus PS3 ATP synthase adopt 'open', 'closed', and 'open' conformations, different from the 'half-closed', 'closed', and 'open' conformations seen in E. coli F₁-ATPase and other bacterial species .

  • These conformational differences suggest species-specific mechanisms of inhibition by subunit ε, which can insert into the α/β interface and force β into different conformations .

• Regulatory Mechanisms:

  • Transcriptional analysis has shown differences in promoter regions and transcription patterns between species.

  • In L. johnsonii and related bacteria, the atp operon is transcribed as both a complete transcript and a shorter transcript, with transcription initiation sites that lack consensus promoter sequences .

• Evolutionary Origins:

  • Phylogenetic analysis demonstrates that the Lactobacillus atpD gene clusters with genera like Listeria, Lactococcus, Streptococcus, and Enterococcus .

  • Higher G+C content and highly biased codon usage compared to the genome average support the hypothesis of horizontal gene transfer in the evolutionary history of Lactobacillus atpD .

• Functional Adaptations:

  • Adaptive changes in the ATP synthase of L. johnsonii likely reflect its specific ecological niche (gut environment) and metabolic capabilities as an obligate homofermentative organism.

  • These adaptations may include optimized activity in the acidic, anaerobic environment of the gastrointestinal tract.

What are the key considerations for designing experiments to study L. johnsonii atpD regulation under different environmental conditions?

When designing experiments to study atpD regulation, researchers should consider the following methodological approaches:

• Environmental Variables to Test:

  • pH: Test range from pH 3.5-7.0, with particular focus on pH 3.5 which has shown maximal induction of ATPase activity .

  • Growth Phase: Compare exponential vs. stationary phase cells, as stationary-phase cells generally show greater acid tolerance .

  • Nutrient Availability: Vary carbon sources and amino acid availability to assess metabolic regulation.

  • Oxygen Levels: L. johnsonii is microaerophilic or anaerobic, so controlling oxygen levels is crucial.

  • Temperature: Test standard growth temperature (37°C) versus stress conditions.

• Expression Analysis Methods:

  • Transcriptional Analysis:

    • qRT-PCR for quantitative assessment of transcript levels

    • Northern blotting for transcript size determination (to identify the complete 7.3 kb transcript and the smaller 4.5 kb transcript)

    • Primer extension to map transcription start sites under different conditions

  • Protein Expression Analysis:

    • Western blotting with specific antibodies against atpD

    • Proteomics approaches (2D-PAGE or LC-MS/MS) to identify co-regulated proteins

• Functional Assays:

  • ATPase Activity Measurement:

    • Colorimetric assays to measure ATP hydrolysis or synthesis rates

    • Measurement of proton translocation using pH-sensitive fluorescent probes

  • Growth and Survival Assays:

    • Acid challenge survival tests

    • Growth rate determination under various conditions

• Genetic Manipulation Approaches:

  • Gene Expression Modulation:

    • Overexpression of atpD to assess effects on acid tolerance

    • Antisense RNA or CRISPR interference to reduce expression

  • Reporter Systems:

    • Promoter-reporter fusions (e.g., lacZ, GFP) to monitor promoter activity

    • Translational fusions to monitor protein expression and localization

• Controls and Statistical Design:

  • Include appropriate reference genes for normalization in qRT-PCR studies

  • Use multiple biological and technical replicates (minimum n=3)

  • Apply appropriate statistical tests (ANOVA with post-hoc tests for multiple conditions)

Studies have shown that the acid inducibility of the atp operon can be verified by slot blot hybridization using RNA isolated from acid-treated cultures, with rapid increases in transcript levels upon exposure to low pH suggesting regulation at the transcriptional level .

How can atpD be used in recombinant protein expression systems for functional studies?

Recombinant L. johnsonii atpD can be utilized in various expression systems for functional studies:

• Heterologous Expression in Model Organisms:

  • E. coli Expression:

    • Use pET expression systems with affinity tags (His, GST) for purification .

    • BL21(DE3) or Rosetta strains are suitable hosts.

    • Expression at lower temperatures (16-25°C) improves proper folding.

  • Bacillus Expression:

    • The Bacillus PS3 ATP synthase has been successfully expressed in E. coli for structural studies .

    • This system could be adapted for L. johnsonii atpD expression.

• Expression in Native Host:

  • Lactobacillus Expression Systems:

    • Xylose-inducible expression systems have been used successfully for protein expression in lactobacilli .

    • The pPG612 vector has been used for successful expression in L. johnsonii .

    • Expression can be verified using western blotting with specific antibodies.

• Functional Complementation:

  • Express L. johnsonii atpD in atpD-deficient strains to assess functional complementation.

  • Compare acid tolerance, growth rates, and ATP synthesis capabilities between wild-type, knockout, and complemented strains.

• Structure-Function Studies:

  • Site-Directed Mutagenesis:

    • Mutate conserved catalytic residues to assess their role in enzyme function.

    • Mutate species-specific amino acid signatures to investigate their functional significance.

  • Domain Swapping:

    • Create chimeric proteins with domains from different species to identify regions responsible for specific functional properties.

• Interaction Studies:

  • Co-expression with Partner Subunits:

    • Co-express atpD with other ATP synthase subunits to study complex assembly.

    • Use pull-down assays to identify interaction partners and assembly intermediates.

  • In vitro Reconstitution:

    • Reconstitute purified atpD with other purified subunits to form functional complexes.

    • Assess ATP synthesis activity of reconstituted complexes.

The successful expression of recombinant L. johnsonii proteins has been demonstrated in multiple studies, including the recent construction of a recombinant L. johnsonii strain expressing bovine GM-CSF, which was verified by western blotting and showed successful biological activity .

What are common challenges in interpreting ATP synthase function across different Lactobacillus species, and how can they be addressed?

Researchers face several challenges when interpreting ATP synthase function across Lactobacillus species:

• Phylogenetic Diversity and Horizontal Gene Transfer:

  • Challenge: Horizontal gene transfer events have influenced the evolution of atpD in Lactobacillus species, complicating phylogenetic interpretations .

  • Solution:

    • Conduct comprehensive phylogenetic analyses incorporating multiple genes.

    • Analyze codon usage patterns and G+C content to identify potential horizontal gene transfer events.

    • Use species-specific amino acid signatures to accurately identify and classify species .

• Metabolic Diversity Among Species:

  • Challenge: Different Lactobacillus species have distinct metabolic capabilities (obligate homofermentative vs. facultative heterofermentative), affecting ATP synthase function .

  • Solution:

    • Conduct comparative studies of ATP synthase activity in species with different metabolic profiles.

    • Consider the whole metabolic network when interpreting ATP synthase function.

    • Use metabolic modeling to predict the role of ATP synthase in different metabolic backgrounds.

• Environmental Adaptation:

  • Challenge: ATP synthase function is influenced by adaptation to specific environments (gut, dairy, plant), making direct comparisons difficult.

  • Solution:

    • Study ATP synthase function under standardized conditions as well as niche-specific conditions.

    • Compare closely related strains isolated from different environments.

    • Correlate functional differences with genomic adaptations.

• Technical Limitations in Activity Measurements:

  • Challenge: Measuring ATP synthase activity accurately, especially in membrane-bound contexts, presents technical challenges.

  • Solution:

    • Use multiple complementary methods to measure activity (ATP production, proton translocation).

    • Develop standardized assays that work across species.

    • Consider both in vitro reconstituted systems and whole-cell approaches.

• Regulatory Differences:

  • Challenge: Regulatory mechanisms controlling ATP synthase expression and activity may differ between species.

  • Solution:

    • Characterize promoter regions and transcription factors across species.

    • Study post-translational modifications affecting enzyme activity.

    • Use comparative transcriptomics and proteomics to identify species-specific regulatory networks.

By addressing these challenges with appropriate methodological approaches, researchers can develop a more comprehensive understanding of ATP synthase function across the diverse Lactobacillus genus.

How can researchers effectively analyze atpD sequence data to identify critical functional domains and species-specific signatures?

Effective analysis of atpD sequence data requires a systematic approach:

• Multiple Sequence Alignment (MSA) and Conservation Analysis:

  • Methodology:

    • Align atpD sequences from diverse Lactobacillus species and other bacteria using MUSCLE or CLUSTALW.

    • Use conservation scoring algorithms (e.g., Jensen-Shannon divergence) to identify highly conserved residues.

    • Generate sequence logos to visualize conservation patterns across the alignment.

  • Interpretation:

    • Highly conserved regions likely represent functionally critical domains.

    • Variable regions may indicate species-specific adaptations or neutral evolution.

• Domain Identification and Functional Annotation:

  • Methodology:

    • Use protein domain databases (Pfam, SMART, InterPro) to identify known functional domains.

    • Map ATP-binding sites, catalytic residues, and interface residues from structural homologs.

    • Predict secondary structure elements using algorithms like PSIPRED.

  • Interpretation:

    • Correlate domain organization with functional differences between species.

    • Identify species-specific insertions or deletions that may affect function.

• Species-Specific Signature Identification:

  • Methodology:

    • Group sequences by species or taxonomic groups.

    • Identify positions that are conserved within groups but differ between groups.

    • Use statistical methods (e.g., mutual information analysis) to identify co-evolving residues.

  • Application:

    • Specific amino acid signatures have been identified in L. johnsonii and L. gasseri strains that distinguish them from other microorganisms .

    • These signatures can be used to design species-specific PCR primers or as targets for restriction enzymes .

• Structural Mapping and Analysis:

  • Methodology:

    • Map sequence variations onto available crystal structures or homology models.

    • Analyze the spatial distribution of conserved and variable residues.

    • Use molecular dynamics simulations to assess the impact of variations on protein dynamics.

  • Interpretation:

    • Surface variations may affect protein-protein interactions or environmental adaptations.

    • Core variations may affect stability, catalytic efficiency, or conformational changes.

• Evolutionary Rate Analysis:

  • Methodology:

    • Calculate synonymous (dS) and non-synonymous (dN) substitution rates.

    • Identify regions under positive or purifying selection.

    • Compare evolutionary rates between lineages.

  • Interpretation:

    • Accelerated evolution in specific lineages may indicate adaptation to new environments.

    • The evolution rate of both enolases seems to be increased in the Lactobacillar branches of the phylogenetic tree, suggesting functional diversification .

This systematic approach has successfully identified 16 conserved signatures specific for the genus Bifidobacterium and multiple amino acid signatures specific to L. johnsonii and L. gasseri strains , demonstrating its effectiveness for functional and taxonomic applications.

What are the key limitations in current understanding of L. johnsonii atpD function, and what research directions might address these gaps?

Current understanding of L. johnsonii atpD function is limited by several factors that future research should address:

• Structural Knowledge Gaps:

  • Current Limitation: No high-resolution structure of L. johnsonii ATP synthase is available.

  • Research Direction:

    • Apply cryo-electron microscopy to determine the structure of the complete ATP synthase complex.

    • Develop improved expression and purification protocols to obtain sufficient quantities of properly assembled complex.

    • Compare structural features with those of other bacterial ATP synthases like the Bacillus PS3 enzyme, which has been successfully imaged .

• Regulatory Mechanisms:

  • Current Limitation: The precise mechanisms controlling atpD expression in response to environmental changes are not fully understood.

  • Research Direction:

    • Identify transcription factors and regulators controlling atp operon expression.

    • Characterize the promoter regions and transcription initiation sites under different conditions.

    • Investigate post-translational modifications affecting enzyme activity.

• Metabolic Integration:

  • Current Limitation: The integration of ATP synthase function with L. johnsonii's unique metabolic capabilities is not completely elucidated.

  • Research Direction:

    • Develop metabolic models incorporating ATP synthase activity.

    • Investigate the coordination between glycolysis, homolactic fermentation, and ATP synthesis.

    • Study energy allocation during stress responses and adaptation.

• Role in Host-Microbe Interactions:

  • Current Limitation: The contribution of ATP synthase to L. johnsonii's probiotic properties and host interactions is poorly understood.

  • Research Direction:

    • Investigate how ATP synthase activity affects colonization and persistence in the gut.

    • Study the role of energy metabolism in competitive fitness against pathogens.

    • Examine whether ATP synthase is targeted by host immune responses.

• Biotechnological Applications:

  • Current Limitation: The potential for engineering atpD for enhanced probiotic or industrial applications is underexplored.

  • Research Direction:

    • Engineer L. johnsonii strains with modified ATP synthase for enhanced acid tolerance.

    • Develop L. johnsonii as a platform for heterologous protein expression using ATP synthase promoters and regulatory elements.

    • Explore the use of L. johnsonii in new therapeutic applications, building on successes like the recombinant L. johnsonii expressing bovine GM-CSF .

Addressing these limitations will require interdisciplinary approaches combining structural biology, biochemistry, molecular genetics, microbial physiology, and computational modeling.

How might advancements in structural biology techniques enhance our understanding of L. johnsonii ATP synthase function?

Recent advancements in structural biology offer exciting opportunities to deepen our understanding of L. johnsonii ATP synthase:

• Cryo-Electron Microscopy (Cryo-EM):

  • Current Status: Cryo-EM has revolutionized structural studies of large macromolecular complexes, including bacterial ATP synthases .

  • Potential Applications:

    • Determine high-resolution structures of L. johnsonii ATP synthase in multiple rotational states.

    • Visualize conformational changes during the catalytic cycle.

    • Identify species-specific structural features that may contribute to unique functional properties.

  • Methodological Approach:

    • Express and purify the complete L. johnsonii ATP synthase complex.

    • Optimize sample preparation conditions for cryo-EM analysis.

    • Use computational classification to identify and reconstruct different conformational states.

• Integrated Structural Biology:

  • Current Status: Combining multiple structural techniques provides complementary information about protein structure and dynamics.

  • Potential Applications:

    • Use X-ray crystallography for high-resolution structures of individual subunits.

    • Apply NMR spectroscopy to study dynamic regions and interactions.

    • Implement hydrogen-deuterium exchange mass spectrometry to map conformational changes.

  • Expected Insights:

    • Detailed understanding of subunit interfaces and interactions.

    • Identification of flexible regions critical for catalysis or regulation.

    • Characterization of ligand-binding sites and their structural changes.

• Molecular Dynamics Simulations:

  • Current Status: Advances in computational power and algorithms allow simulation of large protein complexes at biologically relevant timescales.

  • Potential Applications:

    • Simulate proton translocation through the F₀ domain.

    • Model conformational changes during ATP synthesis and hydrolysis.

    • Predict the effects of mutations on structure and function.

  • Integration with Experimental Data:

    • Validate simulations with experimental structural data.

    • Use simulation predictions to guide mutagenesis experiments.

    • Develop mechanistic models incorporating structural and functional data.

• In situ Structural Biology:

  • Current Status: Methods for studying macromolecular structures in their native cellular environment are rapidly developing.

  • Potential Applications:

    • Use cryo-electron tomography to visualize ATP synthase in intact L. johnsonii cells.

    • Apply correlative light and electron microscopy to link structural and functional information.

    • Implement proximity labeling methods to map the ATP synthase interactome.

  • Expected Insights:

    • Understanding of ATP synthase organization in the bacterial membrane.

    • Identification of interaction partners in vivo.

    • Characterization of structural adaptations to different environmental conditions.

The application of these advanced structural biology techniques to L. johnsonii ATP synthase would build on previous successes with other bacterial ATP synthases, such as the Bacillus PS3 enzyme, which has been imaged by cryo-EM in three rotational states, revealing important insights into mechanism and regulation .

How can engineered forms of recombinant L. johnsonii with modified atpD contribute to probiotic applications?

Engineered forms of L. johnsonii with modified atpD hold significant potential for enhanced probiotic applications:

• Improved Acid Tolerance:

  • Approach: Engineer atpD to enhance proton pumping efficiency or modify its regulation.

  • Potential Benefits:

    • Increased survival during gastric transit

    • Enhanced persistence in the acidic gut environment

    • Improved stability in fermented food products

  • Experimental Design:

    • Target conserved residues in the proton channel or regulatory domains.

    • Use directed evolution to select variants with enhanced acid tolerance.

    • Test survival in simulated gastric conditions and in vivo colonization.

• Enhanced Stress Resistance:

  • Approach: Modify atpD expression regulation to improve response to environmental stresses.

  • Potential Benefits:

    • Better survival during processing and storage

    • Increased resilience against bile salts and digestive enzymes

    • Improved competition against pathogens

  • Implementation:

    • Engineer promoter regions to optimize expression under stress conditions.

    • Develop stress-responsive expression systems using ATP synthase regulatory elements.

• Engineered Therapeutic Delivery Platforms:

  • Approach: Use recombinant L. johnsonii with modified atpD as a platform for delivering therapeutic proteins.

  • Proof of Concept:

    • Recombinant L. johnsonii expressing bovine GM-CSF has demonstrated protective effects against postpartum endometritis by reducing inflammatory cytokines .

  • Potential Applications:

    • Targeted delivery of immunomodulatory factors

    • Production of anti-inflammatory molecules in situ

    • Secretion of antimicrobial peptides against pathogens

• Metabolic Engineering for Enhanced Functionality:

  • Approach: Coordinate atpD modifications with other metabolic engineering strategies.

  • Potential Benefits:

    • Optimized production of beneficial metabolites

    • Reduced formation of undesirable compounds

    • Enhanced competitive fitness in the gut

  • Design Considerations:

    • Balance energy production with metabolic output.

    • Consider the impact on growth rate and competitive fitness.

    • Optimize for specific host environments or conditions.

• Biomarker and Biosensor Development:

  • Approach: Use atpD as a reporter or sensing element for environmental conditions.

  • Applications:

    • Development of biosensors for gut pH or inflammation

    • Real-time monitoring of probiotic activity

    • Strain-specific tracking in clinical studies

The successful engineering of L. johnsonii expressing bovine GM-CSF demonstrates the feasibility of using this species as a delivery platform for therapeutic proteins . This approach could be extended to develop customized probiotics with enhanced functional properties for specific health applications.

What is the potential for using L. johnsonii atpD in synthetic biology applications?

L. johnsonii atpD offers several promising avenues for synthetic biology applications:

• Minimal ATP Synthase Design:

  • Concept: Engineer simplified versions of ATP synthase with reduced complexity but retained functionality.

  • Methodology:

    • Identify essential structural and functional elements through systematic mutagenesis.

    • Design minimal synthetic constructs incorporating only essential components.

    • Test functionality in heterologous expression systems and reconstituted membranes.

  • Potential Applications:

    • Development of energy-generating modules for synthetic cells

    • Creation of simplified bioenergetic systems for educational purposes

    • Design of highly efficient ATP-generating systems for biotechnology

• Modular Energy Systems:

  • Concept: Develop interchangeable modules based on atpD and other ATP synthase components.

  • Design Principles:

    • Standardize interfaces between subunits to allow plug-and-play functionality.

    • Create libraries of variant modules with different properties (efficiency, pH optima, etc.).

    • Design synthetic regulatory elements to control expression and assembly.

  • Applications:

    • Custom-designed energy systems for specific industrial processes

    • Adaptive energy modules responsive to environmental conditions

    • Scalable bioenergetic systems for synthetic biology applications

• Synthetic Metabolic Circuits:

  • Approach: Integrate modified atpD into synthetic metabolic pathways.

  • Design Considerations:

    • Balance ATP production with consumption in synthetic pathways.

    • Coordinate expression of ATP synthase with other metabolic components.

    • Optimize energy efficiency for desired outputs.

  • Potential Applications:

    • Production of high-value compounds requiring precise energy balance

    • Development of self-regulating metabolic systems

    • Creation of artificial ecosystems with defined energy dynamics

• Biosensor Development:

  • Concept: Use atpD as a component of biosensors for environmental monitoring.

  • Mechanism:

    • Couple ATP synthesis or hydrolysis to detectable outputs (fluorescence, electrical signals).

    • Engineer atpD variants responsive to specific environmental conditions.

    • Develop cell-free systems incorporating purified atpD for rapid sensing.

  • Applications:

    • pH sensors for industrial processes or medical diagnostics

    • Energy state monitors for biotechnology applications

    • Environmental toxicity detection systems

• Cross-Kingdom Energy Transfer Systems:

  • Concept: Develop hybrid energy systems combining components from different organisms.

  • Approach:

    • Create chimeric ATP synthases combining bacterial and mitochondrial elements.

    • Test functionality in different cellular contexts and membrane environments.

    • Optimize for specific applications requiring unique properties.

  • Potential Applications:

    • Enhanced energy production in biotechnology hosts

    • Development of novel bioelectronic interfaces

    • Creation of artificial symbionts with optimized energy exchange

The successful expression of functional ATP synthase from Bacillus PS3 in E. coli for structural studies demonstrates the feasibility of heterologous expression of ATP synthase components, providing a foundation for these synthetic biology applications.