Recombinant Lactobacillus plantarum ATP synthase epsilon chain (atpC)

What is the ATP synthase epsilon chain (atpC) in Lactobacillus plantarum and what is its biological function?

The ATP synthase epsilon chain (atpC) is a critical subunit of the F₁F₀-ATP synthase complex in Lactobacillus plantarum. This protein plays a dual role in bacterial energy metabolism:

• Regulation of ATP hydrolysis activity: The epsilon subunit functions as an inhibitor of ATP hydrolysis, particularly important for preventing wasteful ATP consumption under low energy conditions .

• Structural integrity maintenance: The epsilon subunit is essential for maintaining proper assembly and function of the ATP synthase complex, contributing to proton impermeability in the membrane-bound enzyme complex .

The epsilon subunit has a unique structure typically consisting of an N-terminal beta sandwich domain and a C-terminal alpha-helical domain. In many bacteria, these C-terminal helices can adopt either an "up" (inhibitory) or "down" (non-inhibitory) conformation depending on cellular ATP concentration, allowing for dynamic regulation of ATP synthesis/hydrolysis .

How does the atpC gene organization in Lactobacillus plantarum compare to other bacterial species?

The atpC gene in Lactobacillus plantarum is part of the highly conserved atp operon (atpBEFHAGDC), which encodes all subunits of the F₁F₀-ATP synthase. This organization is found across many lactic acid bacteria, though with some variations:

The atp operon is considered highly conserved among eubacteria and has been used as a molecular marker alternative to the 16S rRNA gene for taxonomic studies . Notably, the size of the transcript containing atpC has been identified as approximately 7.3 kb (corresponding to the entire atp operon) and 4.5 kb (corresponding to the atpC, atpD, atpG, and atpA genes) based on Northern blot analysis in related bacteria .

What expression systems are most effective for producing recombinant L. plantarum ATP synthase epsilon chain?

For successful expression of recombinant L. plantarum ATP synthase epsilon chain, several expression systems have shown effectiveness:

Escherichia coli expression system:

• Commonly used for initial expression studies due to high yield and ease of genetic manipulation

• The recombinant protein can be solubilized in 8 M urea and refolded by direct dilution into buffer containing ethanol and glycerol to obtain biologically active epsilon subunit

• Requires optimization of codon usage for effective expression

Lactobacillus expression systems:

• Homologous expression in L. plantarum using vectors such as pWCF has demonstrated success for various recombinant proteins

• Signal peptides like Lp_2145, Lp_0373, and Lp_AmyA have shown higher recombinant protein yields in L. plantarum

• For antibiotic-free selection, asd gene-deficient E. coli (E. coli χ6212) as plasmid donor and alr gene deletion L. plantarum strain NC8Δ as host strain can be used

Mammalian cell expression:

• For specific applications requiring post-translational modifications

• Has been successfully used for expressing recombinant Lactobacillus casei ATP synthase epsilon chain

What are the optimal conditions for solubilization and refolding of recombinant ATP synthase epsilon chain?

Based on research with related ATP synthase epsilon subunits, the following protocol has proven effective:

Solubilization Protocol:

• Express the recombinant protein in E. coli or appropriate expression system

• Lyse cells and isolate inclusion bodies if protein is insoluble

• Solubilize the protein in 8 M urea buffer (pH 8.0)

• For refolding, dilute directly into buffer containing:

  • 10% ethanol (v/v)

  • 20% glycerol (v/v)

  • 50 mM Tris-HCl (pH 8.0)

  • 0.1 mM EDTA

• Allow refolding at room temperature for 1-2 hours

Storage Conditions:

• For short-term storage: 4°C for up to one week

• For long-term storage: Add glycerol to a final concentration of 40-50% and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as this may compromise protein stability and activity

The refolded protein should be assessed for proper folding and biological activity by examining its ability to inhibit the ATPase activity of epsilon-deficient ATP synthase complexes, which serves as a functional assay for correctly folded protein .

How can site-directed mutagenesis be used to study the structure-function relationship of L. plantarum atpC?

Site-directed mutagenesis is a powerful approach to understand the critical functional domains and residues in the ATP synthase epsilon chain:

Key Experimental Approaches:

• N-terminal versus C-terminal modifications:

  • N-terminal truncations generally have more profound effects on epsilon function than C-terminal deletions

  • Design mutations targeting the beta sandwich domain (N-terminus) to study effects on ATP synthase assembly

  • Design mutations in the C-terminal alpha-helical region to study regulatory functions

• Critical residue substitutions:

  • Target conserved residues like histidine-37, which when substituted with arginine can uncouple ATPase inhibition from the restoration of proton impermeability

  • Serine/threonine residues in the C-terminus are potential targets, as these hydroxylated amino acids may be important in epsilon-CF1 interactions

• Functional assays to evaluate mutants:

  • ATPase activity inhibition assay: Measures the ability of recombinant epsilon to inhibit ATPase activity of soluble and membrane-bound F1-ATPase deficient in epsilon

  • Proton impermeability assay: Evaluates the ability of epsilon to restore proton impermeability to thylakoid membranes reconstituted with F1-deficient in epsilon

  • ATP binding assay: Assesses changes in ATP binding capacity, particularly important for mutations in the ATP binding motif I(L)DXXRA

Example Mutation Strategy Table:

What methods are most effective for assessing the biological activity of recombinant L. plantarum ATP synthase epsilon chain?

To comprehensively evaluate the biological activity of recombinant ATP synthase epsilon chain, multiple complementary approaches should be employed:

In vitro Functional Assays:

• ATPase Inhibition Assay:

  • Add purified recombinant epsilon to epsilon-deficient F1-ATPase or CF1

  • Measure ATP hydrolysis activity using a coupled enzyme assay (pyruvate kinase and lactate dehydrogenase) with NADH oxidation monitored at 340 nm

  • Calculate percent inhibition compared to controls

• Proton Impermeability Restoration:

  • Reconstitute thylakoid membranes with CF1 deficient in epsilon

  • Add recombinant epsilon protein

  • Measure proton impermeability using pH-sensitive fluorescent dyes or by monitoring light-dependent pH changes

• ATP Binding Analysis:

  • Isothermal titration calorimetry (ITC) to measure direct binding affinities

  • Fluorescence-based assays using ATP analogs like TNP-ATP

  • Surface plasmon resonance (SPR) to detect real-time binding kinetics

Structural Validation:

• Circular Dichroism (CD) Spectroscopy:

  • Assess secondary structure content and proper folding

  • Monitor conformational changes in response to ATP binding

  • Compare with native protein isolated from L. plantarum

• Differential Scanning Calorimetry (DSC):

  • Evaluate thermal stability of the recombinant protein

  • Compare stability in presence/absence of ATP

• Limited Proteolysis:

  • Determine domain organization and flexibility

  • Compare proteolytic patterns with and without ATP

Reconstitution Experiments:

Reconstitute the purified recombinant epsilon into ATP synthase complexes deficient in the epsilon subunit to assess restoration of:

• ATP synthesis activity

• Regulation of ATP hydrolysis

• Response to changes in ATP/ADP ratio

• Proton pumping efficiency

How does the structure of L. plantarum ATP synthase epsilon chain compare to those from other bacteria, and what implications does this have for its function?

The ATP synthase epsilon chain structure varies somewhat across bacterial species, with important functional consequences:

Structural Comparisons:

The structural differences between Bacillus PS3 and E. coli epsilon subunits are particularly informative. In Bacillus PS3, the C-terminal helices adopt an ATP-dependent "up" conformation, while in E. coli, this conformation is maintained regardless of ATP concentration . This explains why auto-inhibition in E. coli does not depend on ATP concentration while in Bacillus PS3 it does.

The epsilon subunit from Bacillus PS3 can maintain the "up" conformation during ATP synthesis, suggesting it selectively blocks ATP hydrolysis without impeding synthesis . This is made possible by the structural arrangement that creates a clash between subunit ε and β when rotating in the direction of ATP hydrolysis, while allowing rotation in the direction of ATP synthesis .

Based on these comparative structures, the L. plantarum epsilon chain likely shares features with other lactic acid bacteria, potentially with specific adaptations that reflect its ecological niche and metabolic requirements.

What are the molecular mechanisms by which the ATP synthase epsilon chain regulates ATP synthesis versus hydrolysis in L. plantarum?

The regulation of ATP synthesis versus hydrolysis by the epsilon chain involves complex molecular mechanisms:

Key Regulatory Mechanisms:

• Conformational Switching:

  • The C-terminal domain of the epsilon subunit can adopt either an "up" (inhibitory) or "down" (non-inhibitory) conformation

  • In Bacillus PS3, this switching is ATP-dependent: low ATP promotes the inhibitory "up" conformation, while high ATP (>1 mM) induces the permissive "down" conformation

  • The "up" conformation blocks rotation of the central stalk in the direction of ATP hydrolysis while permitting rotation for ATP synthesis

• Interactions with F1 Catalytic Subunits:

  • The epsilon subunit interacts with the α/β interface in the F1 sector

  • In the "up" conformation, it forces the β subunit into an open conformation at the catalytic site (specifically at the βDP position in Bacillus PS3), preventing ATP hydrolysis

  • This structural arrangement creates an asymmetric effect on the three catalytic sites of F1, selectively inhibiting hydrolysis without blocking synthesis

• ATP Binding and Sensing:

  • The ATP binding motif I(L)DXXRA in the epsilon subunit (identified in Bacillus PS3) works together with three arginine and one glutamate residues to recognize ATP

  • ATP binding induces structural changes affecting the C-terminal domain conformation

  • This serves as a molecular sensor of cellular energy status, preventing wasteful ATP hydrolysis when ATP levels are low

Proposed Regulatory Model for L. plantarum:

Based on data from related bacteria, the L. plantarum epsilon subunit likely functions as an ATP-sensing regulatory switch. Under low ATP conditions, it adopts the inhibitory conformation to prevent wasteful ATP hydrolysis. When proton motive force is sufficient and ATP synthesis is favorable, the epsilon subunit allows rotation in the synthesis direction while still blocking hydrolysis.

This directional selectivity is critical for L. plantarum's energy metabolism, especially during transitions between fermentative growth and stress conditions where ATP conservation becomes essential.

How can cross-species comparisons of the ATP synthase epsilon chain inform our understanding of ATP synthase evolution and adaptation?

Cross-species comparisons of the ATP synthase epsilon chain provide valuable insights into the evolution and adaptation of this crucial enzyme:

Evolutionary Conservation and Divergence:

The ATP synthase epsilon chain shows both highly conserved regions and species-specific adaptations:

• Functional Domain Conservation:

  • The N-terminal beta sandwich domain is generally more conserved across species, reflecting its critical role in assembly and structural integrity

  • The ATP-binding motif I(L)DXXRA appears to be conserved in thermophilic bacteria like Bacillus PS3

• Species-Specific Adaptations:

  • C-terminal regions show more variability, with species-specific sequences that may reflect adaptation to different environmental conditions

  • Regulatory mechanisms differ between species: ATP-dependent regulation in Bacillus PS3 versus ATP-independent in E. coli

  • The six amino acids at the C-terminus of spinach epsilon (with four being serine or threonine) represent a region of significant mismatch with pea epsilon and affect ATPase inhibition potency

Phylogenetic Implications:

The atp operon, including atpC, has been used as a molecular marker alternative to 16S rRNA for taxonomic studies . Phylogenetic analysis using atpD (which is in the same operon as atpC) has revealed interesting patterns:

• Lactobacillus atpD genes cluster with genera Listeria, Lactococcus, Streptococcus, and Enterococcus

• Higher G+C content and biased codon usage in some species suggest potential horizontal gene transfer events

Adaptation to Environmental Niches:

Different bacteria have adapted their ATP synthase regulation to suit their ecological niches:

• Thermophilic Adaptation:

  • Bacillus PS3, a thermophile, shows unique ATP-binding properties in its epsilon subunit that may reflect adaptation to high-temperature environments

• Acid Stress Response:

  • In B. lactis DSM 10140, the atp operon shows acid inducibility, with rapid increases in transcript levels upon exposure to low pH

  • This suggests adaptation of ATP synthase regulation to help bacteria cope with acid stress, which would be particularly relevant for lactic acid bacteria like L. plantarum

• Energy Conservation Strategies:

  • The different regulatory mechanisms (ATP-dependent versus independent) likely reflect different energy conservation strategies

  • Organisms like L. plantarum that frequently encounter energy-limited environments may have evolved more sensitive ATP-dependent regulatory mechanisms

What are the main challenges in producing high-yield, properly folded recombinant L. plantarum ATP synthase epsilon chain?

Researchers face several technical challenges when producing the recombinant ATP synthase epsilon chain:

Expression Challenges and Solutions:

Purification Strategies:

• For Denatured-Refolded Protein:

  • Solubilize in 8 M urea

  • Apply to appropriate affinity column under denaturing conditions

  • Perform on-column refolding with decreasing urea gradient

  • Elute and further refold in buffer containing ethanol and glycerol

• For Soluble Expression:

  • Use mild cell lysis methods

  • Implement two-step purification (affinity chromatography followed by size exclusion)

  • Add stabilizing agents (glycerol, reducing agents) to all buffers

  • Consider native purification from L. plantarum as reference standard

• Quality Control Metrics:

  • 85% purity by SDS-PAGE analysis

  • Consistent secondary structure confirmed by circular dichroism

  • Biological activity comparable to native protein isolated from L. plantarum

  • Long-term stability at -20°C or -80°C with 40-50% glycerol

How can structural studies of the L. plantarum ATP synthase epsilon chain best be approached?

Structural characterization of the L. plantarum ATP synthase epsilon chain requires a multi-technique approach:

Recommended Structural Biology Approaches:

• X-ray Crystallography:

  • Optimal for high-resolution structures (1.5-2.5 Å)

  • Crystallization conditions based on successful approaches with related epsilon subunits:

    • Screen with 15-25% PEG 3350/4000, pH 6.5-8.0

    • Add ATP (1-5 mM) to stabilize specific conformations

    • Consider crystallization with binding partners (γ subunit fragment)

  • Example: Crystal structure of ATP-bound epsilon subunit from Bacillus PS3 was determined at 1.9 Å resolution

• Cryo-Electron Microscopy (Cryo-EM):

  • Particularly valuable for visualizing the epsilon subunit in the context of the complete ATP synthase

  • Can reveal different rotational states and conformational changes

  • Example: Cryo-EM of Bacillus PS3 ATP synthase revealed three rotational states at 3.0-3.2 Å resolution

  • Sample preparation is critical: ideally purify intact ATP synthase complexes from L. plantarum

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Excellent for studying dynamics and conformational changes

  • Particularly useful for analyzing ATP-dependent conformational switching

  • Can determine solution structures of the C-terminal domain and measure relaxation parameters

  • Requires isotopic labeling (¹⁵N, ¹³C) of recombinant protein

• Small-Angle X-ray Scattering (SAXS):

  • Provides lower-resolution structural information in solution

  • Useful for studying conformational changes upon ATP binding

  • Can complement higher-resolution techniques

Integrated Structural Biology Workflow:

• Express and purify highly homogeneous recombinant protein

• Perform initial characterization by CD and SAXS

• Attempt crystallization for high-resolution structure

• Use NMR to study dynamics and ATP-induced conformational changes

• If possible, visualize in the context of the complete ATP synthase by cryo-EM

• Integrate all structural data to develop a complete model of function

How can genetic manipulation of the L. plantarum atpC gene be used to engineer bacteria with modified bioenergetic properties?

Genetic manipulation of the L. plantarum atpC gene offers opportunities to create strains with altered bioenergetic characteristics:

Genetic Engineering Strategies:

• Modulating ATP Synthesis/Hydrolysis Balance:

  • Create point mutations in the ATP binding motif to alter ATP sensitivity

  • Engineer C-terminal truncations to modify inhibitory function

  • Introduce mutations that mimic the "down" conformation to reduce inhibition of ATP hydrolysis

• Cross-Species Chimeric Approaches:

  • Replace the L. plantarum epsilon C-terminal domain with that from Bacillus PS3 or E. coli to transfer their regulatory properties

  • Generate chimeras with chloroplast epsilon to study functional convergence

• Site-Directed Mutagenesis Targets:

  • Target the positions equivalent to histidine-37 in spinach chloroplast ATP synthase, which when mutated to arginine uncouples ATPase inhibition from proton impermeability

  • Modify serine/threonine residues in the C-terminus that may be important for interactions with the F1 complex

Potential Applications and Outcomes:

Experimental Design Considerations:

• Use antibiotic-free selection systems based on aspartic acid-β-semialdehyde dehydrogenase (asd) gene and alanine racemase (alr) gene as screening markers

• Employ the pWCF expression vector system, which has been successfully used for recombinant protein expression in L. plantarum

• Confirm genetic modifications by sequencing and validate phenotypic changes through:

  • Measurement of cellular ATP levels

  • Growth rate analysis under different conditions

  • Acid stress tolerance testing

  • Analysis of fermentation products

• For implementation in food-grade systems, ensure all genetic modifications use food-grade selection markers and vectors

How can recombinant L. plantarum ATP synthase epsilon chain be used to study bacterial bioenergetics?

Recombinant L. plantarum ATP synthase epsilon chain serves as a valuable tool for investigating bacterial bioenergetics:

Research Applications:

• Mechanistic Studies of ATP Synthesis Regulation:

  • Use purified recombinant epsilon to reconstitute ATP synthase complexes with defined subunit composition

  • Manipulate epsilon concentration to study dose-dependent effects on ATP synthesis/hydrolysis

  • Compare effects of epsilon from different bacterial species on the same ATP synthase complex

• Investigation of Energy Coupling Mechanisms:

  • Study how the epsilon subunit affects the coupling of proton translocation to ATP synthesis

  • Analyze the role of epsilon in preventing proton leakage, which is critical for maintaining the proton motive force

  • Examine how epsilon contributes to the efficiency of energy conversion

• Analysis of Bacterial Adaptation to Energy Stress:

  • Use recombinant epsilon with reporter systems to monitor conformational changes under different energy conditions

  • Compare epsilon behavior from bacteria adapted to different environments (acidic, alkaline, energy-limited)

  • Study post-translational modifications of epsilon that might occur during stress responses

Experimental Approaches:

• In vitro Reconstitution Systems:

  • Purify individual ATP synthase components and reconstitute with varying amounts of recombinant epsilon

  • Measure ATP synthesis/hydrolysis activities under defined conditions

  • Analyze proton pumping using pH-sensitive fluorescent dyes

• Single-Molecule Studies:

  • Attach fluorescent labels to specific residues on recombinant epsilon

  • Use FRET (Förster Resonance Energy Transfer) to monitor conformational changes in real-time

  • Employ optical trapping to study the mechanical aspects of ATP synthase rotation and how epsilon affects this process

• Comparative Biochemistry:

  • Compare the regulatory properties of epsilon from L. plantarum with those from other lactic acid bacteria and more distantly related species

  • Analyze evolution of regulatory mechanisms across bacterial lineages

  • Identify adaptations specific to the ecological niche of L. plantarum

What insights can be gained from studying the interaction between the ATP synthase epsilon chain and other subunits of the ATP synthase complex?

Studying interactions between the epsilon chain and other ATP synthase subunits provides critical insights into enzyme function:

Key Interaction Partners and Their Significance:

• Epsilon-Gamma Subunit Interactions:

  • The epsilon subunit interacts extensively with the gamma subunit in the central stalk

  • These interactions are crucial for the transmission of conformational changes during rotary catalysis

  • In E. coli, a 10-residue loop allows the second α-helix of epsilon to interact with subunit γ, potentially stabilizing the "up" conformation

  • Studying these interactions can reveal how mechanical energy is transmitted during ATP synthesis/hydrolysis

• Epsilon-Beta Subunit Interactions:

  • The epsilon subunit in the "up" conformation inserts into the α/β interface

  • This insertion forces β into specific conformations (e.g., "open" conformation at the βDP position in Bacillus PS3)

  • Understanding these interactions reveals how epsilon selectively inhibits ATP hydrolysis while allowing ATP synthesis

• Epsilon-c-Ring Interactions:

  • The epsilon subunit connects the F₁ and F₀ sectors of ATP synthase

  • These interactions are critical for coupling proton translocation to ATP synthesis

  • Analyzing these interactions provides insights into the efficiency of energy conversion

Methodological Approaches:

• Cross-linking Studies:

  • Use chemical cross-linking followed by mass spectrometry to identify interaction sites

  • Apply site-specific cross-linkers to map the interaction surfaces in detail

  • Compare cross-linking patterns with and without ATP to detect conformational changes

• Co-immunoprecipitation and Pull-down Assays:

  • Use tagged recombinant epsilon to pull down interacting subunits

  • Identify interaction partners under different conditions (ATP, ADP, pH, etc.)

  • Quantify binding affinities using surface plasmon resonance or isothermal titration calorimetry

• Hybrid Structural Approaches:

  • Combine cryo-EM of the full complex with higher-resolution structures of individual components

  • Use computational modeling to predict interaction interfaces

  • Validate predictions through site-directed mutagenesis of key residues

Research Implications:

Understanding these interactions has implications for:

• Developing antimicrobial compounds targeting ATP synthase

• Engineering ATP synthases with improved efficiency

• Understanding bacterial adaptation to environmental stressors

• Elucidating evolutionary relationships between different bacterial ATP synthases

What are the potential applications of recombinant L. plantarum expressing modified ATP synthase epsilon chain in probiotic research?

Recombinant L. plantarum strains with modified ATP synthase epsilon chains offer unique opportunities for probiotic research:

Potential Applications in Probiotic Research:

• Enhanced Stress Tolerance:

  • Engineering L. plantarum with modified epsilon subunits could improve survival under gastrointestinal conditions

  • Strains with altered ATP synthesis regulation might better withstand acid stress, enhancing gastric transit survival

  • Increased energy efficiency could improve colonization potential

• Metabolic Engineering for Therapeutic Benefits:

  • Modifying ATP synthesis efficiency could alter metabolic end-product profiles

  • Engineered strains might produce higher levels of beneficial metabolites (short-chain fatty acids, specific vitamins)

  • Control of ATP/ADP ratio could influence production of immunomodulatory compounds

• Delivery Vehicles for Biotherapeutics:

  • L. plantarum is already being explored as a vehicle for delivering therapeutic proteins to mucosal surfaces

  • Strains with optimized bioenergetics could provide more efficient production and delivery of therapeutic proteins

  • The existing research on recombinant L. plantarum for vaccine delivery provides a foundation for this application

Research Approaches:

• Rational Design of Bioenergetically Optimized Probiotics:

  • Identify epsilon modifications that enhance survival under specific stress conditions

  • Engineer strains with targeted changes to ATP synthesis regulation

  • Test survival and colonization potential in in vitro and in vivo models

• Integration with Other Probiotic Traits:

  • Combine engineered ATP synthase with other beneficial modifications (e.g., enhanced adhesion, immunomodulatory properties)

  • Develop multi-functional probiotic strains with optimized energy metabolism and therapeutic capabilities

  • Use systems biology approaches to predict optimal combinations of modifications

• Safety and Efficacy Assessment:

  • Evaluate genetic stability of engineered strains over multiple generations

  • Assess potential transfer of modified genes to gut microbiota

  • Compare immunomodulatory effects of engineered strains with wild-type L. plantarum

Practical Considerations:

For probiotic applications, it's essential to use food-grade genetic modification systems:

• Antibiotic-free selection systems based on aspartic acid-β-semialdehyde dehydrogenase (asd) gene and alanine racemase (alr) gene as screening markers

• Self-limiting systems that prevent environmental spread of genetically modified organisms

• Thorough safety assessment before human studies