Recombinant Pediococcus pentosaceus ATP synthase subunit b (atpF)

What expression systems work best for recombinant production of P. pentosaceus atpF?

Successful expression of recombinant P. pentosaceus atpF requires careful consideration of expression systems. Based on research with similar ATP synthase subunits:

For optimal expression:

• Use E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3))

• Culture at lower temperatures (16-25°C) to promote proper folding

• Use moderate inducer concentrations (0.1-0.5 mM IPTG)

• Consider synthetic gene construction with optimized codon usage

A method demonstrated for ATP synthase subunits involves induction with 1.0 mM IPTG for 30 minutes, followed by cell disruption in lysis buffer (20 mM Tris-HCl pH 8.0 with protease inhibitors) using lysozyme treatment and sonication .

How does the genomic context of atpF differ between P. pentosaceus strains?

Comparative genomic analysis across P. pentosaceus strains reveals important insights about atpF conservation and variability:

P. pentosaceus typically has a genome size of approximately 1.8 Mb with a GC content of 37-38% . The genomic analyses reveal:

• The atpF gene (PEPE_1321 in the reference strain ATCC 25745) is part of the ATP synthase operon

• Analysis of 176 P. pentosaceus genomes shows conservation of ATP synthase genes within the core genome

• The core genome of P. pentosaceus includes genes encoding proteins related to translation, ribosomal structure, and signal transduction mechanisms

While ATP synthase genes are generally conserved, the genetic diversity between strains relates mainly to:

• Carbohydrate metabolism

• Horizontally transferred DNA

• Prophage sequences

• Bacteriocins encoded on plasmids

The gene encoding ATP synthase subunit b appears to be chromosomally located rather than on plasmids, as research has identified strains like IMI 507025 with no plasmids but complete ATP synthase machinery .

What purification strategies are most effective for recombinant P. pentosaceus atpF?

Purifying recombinant P. pentosaceus ATP synthase subunit b requires specialized approaches due to its membrane protein nature:

Recommended Purification Protocol:

• Fusion Tag Selection:

  • His-tag is widely used as seen in other ATP synthase subunit b proteins

  • MBP-tag can enhance solubility while providing affinity purification

• Membrane Extraction:

  • Two-phase extraction beginning with mild detergent (0.5% DDM)

  • Sequential extraction with increasing detergent strength

• Buffer Composition:

  • Tris-based buffer with glycerol for stability

  • Detergent concentration above CMC but minimized to prevent protein destabilization

• Storage Conditions:

  • Store at -20°C or -80°C in buffer containing 50% glycerol

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be maintained at 4°C for up to one week

Quality assessment should include SDS-PAGE (target >90% purity), western blotting, and mass spectrometry to confirm protein identity and integrity.

How does recombinant P. pentosaceus atpF contribute to understanding bacterial energy metabolism?

Recombinant P. pentosaceus ATP synthase subunit b provides a valuable tool for investigating bacterial bioenergetics:

Research Applications:

• Structure-Function Studies:

  • Site-directed mutagenesis of specific residues to map functional domains

  • Investigation of how different regions contribute to ATP synthase assembly

  • Analysis of the coupling mechanism between proton translocation and ATP synthesis

• Comparative Bioenergetics:

  • The c-subunit ring stoichiometry determines the H⁺/ATP ratio, which varies between organisms (from c₁₀ to c₁₅)

  • Coupling ratio (ions transported:ATP generated) ranges from 3.3 to 5.0 across species

  • Studies with recombinant subunits help elucidate species-specific adaptations

• Reconstitution Experiments:

  • Assembly of functional ATP synthase complexes in liposomes

  • Controlled studies of proton translocation under defined conditions

  • Analysis of energy coupling efficiency

• Antimicrobial Development:

  • Screening for molecules that specifically target bacterial ATP synthase

  • Structure-based design of inhibitors that exploit differences between bacterial and human ATP synthases

These approaches contribute to fundamental understanding of bacterial energy metabolism and may identify novel antibacterial targets.

What technical challenges must be overcome when expressing P. pentosaceus atpF in heterologous systems?

Expressing P. pentosaceus atpF in heterologous systems presents several challenges that require specific strategies to overcome:

Challenge 1: Codon Usage Bias

P. pentosaceus has a low GC content (37-38%) compared to E. coli (50-51%), leading to potential translation inefficiencies.

Solution:

• Synthesize codon-optimized genes as demonstrated for ATP synthase subunits

• Consider using the overlapping oligonucleotide approach, which allows for complete codon optimization

• Alternatively, use E. coli strains supplemented with rare tRNAs (Rosetta)

Challenge 2: Membrane Protein Solubility

ATP synthase subunit b contains hydrophobic regions that often lead to aggregation.

Solution:

• Use fusion partners (MBP, SUMO) that enhance solubility

• Express at reduced temperatures (16-20°C)

• Include mild detergents during extraction and purification

• Consider membrane-mimetic systems (nanodiscs, amphipols) for structural studies

Challenge 3: Proper Folding

Membrane proteins often require specific lipid environments for correct folding.

Solution:

• Optimize expression conditions (temperature, inducer concentration)

• Co-express with molecular chaperones (GroEL/GroES)

• Consider cell-free expression systems with added phospholipids

Challenge 4: Functional Validation

Confirming that recombinant atpF maintains native structure and function.

Solution:

• Use circular dichroism (CD) spectroscopy to verify secondary structure

• Perform binding assays with partner ATP synthase subunits

• Attempt functional complementation in bacterial strains with inactivated atpF

Critical Regions for Mutagenesis Studies:

• Transmembrane Domain (N-terminal region)

  • Mutations disrupting membrane anchoring affect assembly and stability

  • Alterations in hydrophobic residues can interfere with integration into the lipid bilayer

  • Changes affecting interaction with subunit a may disrupt the proton path

• Dimerization Interface (C-terminal domain)

  • Mutations disrupting dimerization destabilize the stator stalk

  • Changes in key residues can weaken the coiled-coil structure

  • This typically results in uncoupling of proton translocation from ATP synthesis

• F₁ Interaction Region (uppermost C-terminal portion)

  • Mutations affecting interaction with δ-subunit compromise the connection to F₁

  • This can lead to decreased efficiency without completely abolishing function

Experimental Approaches:

For systematic analysis of atpF mutations:

• Generate point mutations using site-directed mutagenesis

• Express recombinant variants and assess structural integrity

• Reconstitute into proteoliposomes with other ATP synthase subunits

• Measure ATP synthesis activity under defined PMF (proton motive force)

• Test complementation ability in atpF-deficient bacterial strains

Mutations in ATP synthase genes typically have pleiotropic effects on bacterial physiology, affecting not only energy generation but also membrane potential maintenance and pH homeostasis.

What is the relationship between P. pentosaceus atpF and the probiotic properties of this bacterium?

The relationship between ATP synthase function and the probiotic properties of P. pentosaceus can be understood through several mechanisms:

Energy Metabolism and Probiotic Functions:

P. pentosaceus exhibits numerous probiotic effects including:

• Antioxidant and cholesterol-reducing properties

• Production of antimicrobial substances (bacteriocins)

• Immune-enhancing capabilities

ATP synthase provides the energy currency required for these beneficial functions:

• Stress Adaptation:
  P. pentosaceus ENM104 contains genes for stress adaptation (e.g., htpX, dnaK, dnaJ for heat stress; ppaC for bile tolerance) . ATP synthase activity supports these energy-dependent stress responses essential for survival in the GI tract.

• Bacteriocin Production:
  P. pentosaceus strains produce various bacteriocins including pediocin-like bacteriocins:

  • Penocin A (encoded by penA)

  • Coagulin A

  • Pediocin PA-1

  • Plantaricin 423

  These antimicrobial peptides require energy for synthesis, processing, and secretion.

• Metabolite Production:
  ATP synthase supports energy-dependent synthesis of:

  • GABA (via glutamate/GABA antiporter, gadC)

  • Vitamins (e.g., riboflavin pathway genes ribU, ribZ, ribF)

• Immunomodulation:
  ATP-dependent processes support production of immunomodulatory factors (e.g., dltA, dltC, dltD) and exopolysaccharides that interact with host immune cells.

Recent research with P. pentosaceus KF159 demonstrates that this strain alleviates atopic dermatitis-like symptoms by modulating Th1/Th2 immune balance and inhibiting IgE production , processes that rely on cellular energy provided by ATP synthase.

How can recombinant atpF be used to develop antibacterial agents specific to pathogenic bacteria?

While P. pentosaceus is beneficial, insights from its ATP synthase structure can inform antibacterial development through several approaches:

Structure-Based Drug Design Strategy:

• Comparative Structural Analysis:

  • Generate high-resolution structures of recombinant P. pentosaceus atpF

  • Compare with atpF from pathogenic bacteria to identify structural differences

  • Map conservation patterns to distinguish essential core regions from variable surfaces

• Binding Site Identification:

  • Identify potential binding pockets present in pathogenic bacteria but absent or structurally different in beneficial bacteria

  • Use computational approaches (molecular docking, MD simulations) to characterize binding site properties

• Selective Inhibitor Design:

  • Design small molecules targeting pathogen-specific binding sites

  • Screen compound libraries against panels of recombinant atpF proteins from different bacteria

  • Optimize lead compounds for selectivity and potency

Experimental Validation Process:

• In vitro Testing:

  • ATP synthesis/hydrolysis assays with reconstituted ATP synthase complexes

  • Growth inhibition assays against panels of pathogenic and beneficial bacteria

  • Resistance development monitoring through serial passage experiments

• Mechanism of Action Studies:

  • Site-directed mutagenesis to confirm binding sites

  • Cryo-EM structures of inhibitor-bound ATP synthase complexes

  • Biochemical characterization of inhibition mechanisms (competitive vs. non-competitive)

This approach could lead to narrow-spectrum antibiotics that selectively target pathogenic bacteria while preserving beneficial microbiota like P. pentosaceus.

What structural adaptations in P. pentosaceus atpF contribute to its environmental resilience?

P. pentosaceus is found in diverse environments including fermented foods and the gastrointestinal tract, suggesting adaptations in its ATP synthase for environmental resilience:

Structural Elements Contributing to Resilience:

• Membrane-Spanning Domain:

  • The N-terminal hydrophobic region of atpF contains specific amino acid patterns that balance membrane anchoring with flexibility

  • Comparative analysis with atpF sequences from other bacteria could reveal adaptations specific to P. pentosaceus

• Stator Stability Features:

  • The C-terminal domain contains residue patterns that form a rigid stator stalk while accommodating stress

  • This region likely includes adaptations that maintain function during pH fluctuations encountered in fermentation environments

• Interaction Interfaces:

  • The interfaces between atpF and other ATP synthase subunits may contain specific adaptations that maintain complex integrity under stress conditions

  • These could include salt bridges and hydrophobic interactions that remain stable across varying environmental conditions

Experimental Evidence of Resilience:

P. pentosaceus exhibits several stress tolerance mechanisms that may relate to ATP synthase adaptation:

• Acid tolerance genes that support survival during fermentation and gastric passage

• Heat stress response proteins (htpX, dnaK, dnaJ) that maintain cellular function during temperature fluctuations

• Bile tolerance mechanisms (e.g., ppaC) that enable survival in the intestinal environment

ATP synthase must remain functional across these stress conditions to supply the energy needed for adaptive responses, suggesting structural adaptations in atpF that maintain ATP synthesis capacity under varying environmental conditions.

How does the c-ring stoichiometry in P. pentosaceus ATP synthase affect its bioenergetic efficiency?

The c-ring stoichiometry is a critical determinant of ATP synthase bioenergetic efficiency:

Relationship Between c-ring Stoichiometry and Bioenergetics:

• Coupling Ratio Determination:

  • The number of c-subunits in the ring (n) determines the H⁺/ATP ratio

  • This ratio equals n/3, as each 360° rotation of the c-ring synthesizes 3 ATP molecules

  • In different organisms, n ranges from 10 to 15, resulting in coupling ratios from 3.3 to 5.0

• The Specific Case of P. pentosaceus:
  While the exact c-ring stoichiometry for P. pentosaceus is not specified in the literature, we can make educated inferences:

  • Based on the low GC content (37-38%) similar to other lactic acid bacteria

  • Probable adaptation to fermentative metabolism with variable energy availability

  • Likely balances ATP yield against physiological PMF range

• Functional Implications:

  • Higher c-ring stoichiometry provides more ATP per rotation but requires higher PMF

  • Lower stoichiometry allows ATP synthesis at lower PMF but with reduced yield

  • The stoichiometry represents an evolutionary adaptation to the organism's ecological niche

Experimental Approaches to Determine c-ring Stoichiometry:

• Isolation and Mass Determination:

  • Isolate intact c-rings through specialized purification

  • Determine mass through techniques like native mass spectrometry

  • Calculate subunit number from total mass and monomeric mass

• Structural Characterization:

  • Electron microscopy of purified c-rings

  • X-ray crystallography to determine exact subunit arrangement

  • Atomic force microscopy to count subunits

• Functional Measurements:

  • H⁺/ATP ratio determination through reconstitution experiments

  • Thermodynamic efficiency calculations based on phosphorylation potential and PMF

Understanding this parameter would provide valuable insights into P. pentosaceus bioenergetic adaptation to its ecological niche.

What controls should be included when studying recombinant P. pentosaceus atpF in vitro?

Rigorous controls are essential when studying recombinant P. pentosaceus ATP synthase subunit b to ensure valid interpretation of results:

Essential Control Experiments:

When performing reconstitution experiments:

• Include liposomes without protein to measure background proton leakage

• Use uncouplers (CCCP) as positive controls for maximum proton permeability

• Include c-subunit-only controls to distinguish full complex activities

These controls help ensure that experimental observations are specifically attributable to properly folded and functional P. pentosaceus atpF rather than experimental artifacts.

How can codon optimization strategies improve recombinant P. pentosaceus atpF expression?

Codon optimization is critical for heterologous expression of P. pentosaceus atpF due to significant differences in codon usage between P. pentosaceus and common expression hosts:

Codon Usage Comparison:

P. pentosaceus has a low GC content of approximately 37-38% , while E. coli has a GC content of 50-51%. This difference leads to codon usage bias that can significantly impact expression efficiency.

What methods can be used to study the interaction between recombinant atpF and other ATP synthase subunits?

Understanding interactions between ATP synthase subunits is crucial for elucidating the complex's structure and function:

Protein-Protein Interaction Methods:

• Co-purification Approaches:

  • Co-expression of atpF with binding partners (e.g., subunit a, subunit δ)

  • Tandem affinity purification to isolate intact subcomplexes

  • Analytical size exclusion chromatography to determine complex formation

• Binding Assays:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics and affinities

  • Microscale Thermophoresis (MST) for label-free interaction analysis

  • Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters

• Crosslinking Studies:

  • Chemical crosslinking with BS3 or EDC to capture transient interactions

  • Photo-crosslinking with genetically incorporated unnatural amino acids

  • Mass spectrometry analysis of crosslinked peptides to map interaction interfaces

• Structural Biology Approaches:

  • Cryo-EM of reconstituted ATP synthase complexes or subcomplexes

  • X-ray crystallography of co-crystallized subunits

  • NMR for mapping interaction surfaces of isolated domains

Experimental Design for P. pentosaceus ATP Synthase:

For studying atpF interactions with other ATP synthase subunits:

• Partner Identification:

  • Express and purify recombinant atpF with an affinity tag

  • Immobilize on appropriate resin

  • Incubate with P. pentosaceus cell lysate

  • Analyze bound proteins by mass spectrometry

• Interface Mapping:

  • Generate truncation variants of atpF to identify minimal binding domains

  • Perform site-directed mutagenesis of conserved residues

  • Measure effects on binding affinity and ATP synthase assembly

• Functional Validation:

  • Reconstitute purified subunits into liposomes

  • Measure proton pumping and ATP synthesis activities

  • Correlate structural findings with functional outcomes

These approaches would provide valuable insights into the assembly and function of P. pentosaceus ATP synthase, potentially revealing species-specific features.

How does the genomic diversity of P. pentosaceus strains affect ATP synthase structure and function?

Genomic analysis of multiple P. pentosaceus strains provides insights into ATP synthase conservation and variation:

Comparative Genomic Analysis:

Analysis of 176 P. pentosaceus genomes reveals patterns relevant to ATP synthase:

• Core vs. Accessory Genome:

  • ATP synthase genes (including atpF) belong to the core genome shared across strains

  • The core genome includes genes encoding proteins related to translation, ribosomal structure, and signal transduction mechanisms

  • This conservation reflects the essential nature of ATP synthase for cellular energy metabolism

• Strain-Specific Variations:

  • While ATP synthase genes are conserved, subtle sequence variations may exist

  • These variations potentially adapt the enzyme to specific ecological niches

  • Different strains show variation in:

    • Stress response capabilities

    • Metabolic pathways

    • Carbohydrate utilization patterns

• Genomic Context:

  • The ATP synthase operon structure appears conserved across strains

  • Genomic regions showing highest variability include mobile genetic elements and strain-specific metabolic pathways

Functional Implications:

The comparative analysis of P. pentosaceus LI05 with food-borne strains (ATCC 25745, SL4, and IE-3) revealed:

• Conserved core metabolic functions including energy generation

• Strain-specific adaptations for environmental stress tolerance

• Variability in exopolysaccharide biosynthesis proteins

These findings suggest that while the essential function of ATP synthase is preserved across strains, subtle variations may fine-tune its performance for specific environmental conditions, potentially affecting:

• Operating pH range

• Temperature optimum

• Regulatory responses to environmental stressors