Recombinant Lactobacillus plantarum ATP synthase subunit b (atpF)

Basic Research Questions

• What is the structural composition of ATP synthase subunit b (atpF) in Lactobacillus plantarum?

  ATP synthase subunit b (atpF) in L. plantarum is a membrane-associated protein with 171 amino acids. The complete sequence is: mLSHLIIGASGLYLGDmLFIGISFIVLMALISVVAWKPITKMMADRADKIANDIDSAQKSRQEASDLADQRRDALSHSRAEASEIVADAKKSGEKQRSSIVADAQNEATQYKQNARKDIEQERQDALKNVQSDVADISVAIATKIIKKQLDPEGQQALINSYIEGLGKHES .

  The protein contains a transmembrane domain in its N-terminal region that anchors it to the membrane, followed by a cytoplasmic domain that interacts with other subunits of the ATP synthase complex. The subunit b is part of the F0 sector of F1F0-ATPase, which forms the membrane-embedded portion responsible for proton translocation across the bacterial membrane .

• How does ATP synthase function in the energy metabolism of Lactobacillus plantarum?

  In L. plantarum, ATP synthase functions as a reversible molecular machine that can either synthesize ATP using the energy from a transmembrane proton gradient or hydrolyze ATP to generate this gradient. Unlike strictly fermentative lactic acid bacteria, L. plantarum exhibits a blended metabolism combining features of respiration and fermentation .

  The process involves:

  • Proton translocation across the membrane-embedded F0 sector (where atpF resides)

  • Rotation of the c-ring in response to proton flow

  • Conformational changes in the F1 sector containing the catalytic sites

  • Synthesis of ATP from ADP and inorganic phosphate

  This mechanism is essential for:

  • Maintaining pH homeostasis in acidic environments

  • Supporting energy metabolism efficiency

  • Enhancing probiotic survival in the gastrointestinal tract

• What distinguishes L. plantarum ATP synthase from other bacterial ATP synthases?

  L. plantarum ATP synthase shows several distinctive features compared to other bacterial homologs:

  • The catalytic β subunits in L. plantarum (like those in Bacillus PS3) adopt 'open', 'closed', and 'open' conformations, differing from the 'half-closed', 'closed', and 'open' conformations seen in E. coli F1-ATPase

  • This conformational difference suggests species-specific differences in inhibition mechanisms by regulatory subunits

  • L. plantarum ATP synthase operates in more acidic environments compared to many other bacteria, with adaptations that allow it to function efficiently at pH values as low as 4.6, such as during Sichuan pickle fermentation

  • The enzyme contributes to L. plantarum's ability to perform extracellular electron transfer (EET), a process that increases intracellular NAD+:NADH ratios and affects fermentation flux

Advanced Research Questions

• How do mutations in ATP synthase affect acid tolerance in L. plantarum, and what methodologies are most reliable for measuring this effect?

  Mutations in ATP synthase components significantly alter acid tolerance in L. plantarum through impaired proton pumping capabilities. Research methodologies to study this relationship include:

  Mutation analysis approach:

  • Site-directed mutagenesis targeting conserved residues (comparable to the Ser-268→Leu-268 mutation that reduced ATPase activity by 43.44% in strain LPM21)

  • Construction of deletion mutants using homologous recombination techniques

  Functional assessment methods:

  • Membrane-bound ATPase activity assays under acidic conditions (pH 4.6) using Nannen and Hutkins methodology

  • Growth curve analysis at different pH values

  • Intracellular pH measurements using fluorescent probes

  • Survival rate determination following acid challenge

  Data interpretation framework:

  Strain	ATPase Activity (U/mg protein)	Reduction vs. Parent (%)	pH Tolerance
  Wild-type	0.356 ± 0.015	—	Reference
  Mutant A	0.336 ± 0.011	5.61	Minimal reduction
  Mutant B	0.201 ± 0.009	43.44	Significant reduction
  Mutant C	0.287 ± 0.013	19.46	Moderate reduction

  For reliable results, researchers should combine these approaches with transcriptomic and proteomic analyses to account for potential compensatory mechanisms that might be activated in response to ATP synthase mutations .

• What are the critical residues in atpF that affect interaction with other ATP synthase subunits, and how can they be experimentally determined?

  Critical residues in atpF that mediate subunit interactions can be identified through a combination of structural and functional approaches:

  Computational methods:

  • Homology modeling based on the cryo-EM structures of related ATP synthases (like the Bacillus PS3 ATP synthase)

  • Molecular dynamics simulations to predict interaction interfaces

  • Conservation analysis across species to identify evolutionarily conserved residues

  Experimental approaches:

  • Alanine scanning mutagenesis targeting predicted interface residues

  • Chemical cross-linking followed by mass spectrometry (XL-MS) to identify proximity relationships

  • FRET (Förster Resonance Energy Transfer) analysis with fluorescently labeled subunits

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify protected regions

  Key residues of interest:
  Based on homology with E. coli ATP synthase, several residues might be particularly important:

  • Transmembrane anchoring residues in the N-terminal region

  • Residues analogous to Arg169 in Bacillus PS3 (corresponding to Arg210 in E. coli), which is critical for proton translocation

  • Residues corresponding to Glu159, Glu178, Ser210, Asp19, Asn173, and Gln217 in Bacillus PS3, which have been identified as functionally important in E. coli

  The experimental validation should involve site-directed mutagenesis of these residues followed by functional assays to assess ATP synthase assembly and activity.

• How does atpF contribute to extracellular electron transfer (EET) in L. plantarum, and what techniques can resolve this mechanistic relationship?

  L. plantarum performs EET through a blended metabolism combining features of respiration and fermentation . The role of atpF in this process can be investigated through:

  Mechanistic investigation methods:

  • Electrochemical techniques: Chronoamperometry, cyclic voltammetry, and differential pulse voltammetry to measure electron transfer rates in wild-type vs. atpF mutants

  • Spectroscopic methods: EPR spectroscopy to detect radical intermediates involved in electron transfer

  • Isotope labeling: 13C labeling to track metabolic fluxes during EET

  Genetic approaches:

  • Construction of atpF deletion or point mutants

  • Complementation studies with wild-type or modified atpF genes

  • Transcriptional fusion reporters to monitor gene expression changes during EET

  Biochemical characterization:

  • Membrane fraction isolation and measurement of electron transport chain component activities

  • Identification of electron carriers that interact with ATP synthase during EET

  • Correlation between NAD+:NADH ratios and ATP synthase activity

  This approach would help determine whether atpF plays a direct role in EET (as part of an electron transport chain) or an indirect role (through effects on proton motive force or energy metabolism) .

Methodological Questions

• What expression systems are most effective for producing functional recombinant L. plantarum ATP synthase subunit b, and how should expression be optimized?

  Optimal expression of functional recombinant L. plantarum atpF requires careful selection of expression systems and optimization strategies:

  Expression host selection:

  • E. coli: Most commonly used, as seen with the recombinant L. fermentum ATP synthase subunit b expression

  • Lactobacillus species: Homologous expression for proper folding and post-translational modifications

  • Cell-free systems: For difficult-to-express membrane proteins

  Vector design considerations:

  • Codon optimization for the selected expression host

  • Addition of affinity tags (His-tag being common) for purification, with consideration of tag position (N or C-terminal) based on membrane topology

  • Inclusion of protease cleavage sites for tag removal

  • Use of inducible promoters for controlled expression

  Expression optimization protocol:

  1. Temperature optimization (typically lower temperatures of 16-25°C for membrane proteins)

  2. Inducer concentration titration

  3. Media composition adjustment (including use of osmolytes or chaperone co-expression)

  4. Expression time optimization (monitoring expression at 2-hour intervals)

  5. Membrane solubilization screening with different detergents (such as glycol-diosgenin/GDN used for ATP synthase purification)

  Purification strategy:

  1. Membrane fraction isolation

  2. Detergent solubilization (testing multiple detergents: DDM, GDN, LMNG)

  3. Affinity chromatography (IMAC for His-tagged proteins)

  4. Size exclusion chromatography to obtain homogeneous protein

  5. Protein quality assessment by SDS-PAGE and Western blotting

  To maintain functionality, consideration of lipid environment during purification and reconstitution is critical for this membrane protein.

• What are the most sensitive methods for assessing the functionality of recombinant L. plantarum ATP synthase subunit b in vitro?

  Evaluating the functionality of recombinant atpF requires assessing both its structural integrity and its ability to function within the ATP synthase complex:

  Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Thermal shift assays to measure protein stability

  • Native PAGE to assess oligomerization state

  • Limited proteolysis to evaluate folding quality

  Functional assays:

  • Reconstitution into liposomes or nanodiscs with other ATP synthase subunits

  • ATP hydrolysis assay using colorimetric phosphate detection (modified Malachite Green assay)

  • Proton translocation measurements using pH-sensitive fluorescent dyes

  • Membrane potential measurements using potential-sensitive dyes

  Interaction studies:

  • Surface plasmon resonance (SPR) to measure binding to other subunits

  • Isothermal titration calorimetry (ITC) for binding thermodynamics

  • Pull-down assays to confirm interactions with partner proteins

  • Microscale thermophoresis (MST) for interaction studies in solution

  Advanced biophysical methods:

  • Single-molecule FRET to monitor conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • Electron microscopy to visualize integration into the ATP synthase complex

  Comparative analysis with wild-type protein and known functional variants should be included as controls in all experiments.

• How can researchers effectively engineer mutations in L. plantarum atpF to study structure-function relationships?

  Engineering mutations in L. plantarum atpF for structure-function studies requires a systematic approach:

  Target selection strategies:

  • Sequence alignment with homologous proteins to identify conserved residues

  • Structure-based identification of residues at subunit interfaces or in functional domains

  • Evolutionary analysis to identify co-evolving residues

  • Molecular modeling to predict critical residues

  Mutation design approaches:

  • Alanine scanning: Systematic replacement with alanine to identify essential residues

  • Conservative substitutions: Replacing with similar amino acids to fine-tune function

  • Non-conservative substitutions: Testing dramatic changes to probe function

  • Domain swapping: Replacing domains with those from other species to test specificity

  Genetic engineering methods:

  • Site-directed mutagenesis for precise single mutations

  • Gibson Assembly or Golden Gate cloning for multiple mutations

  • CRISPR-Cas9 genome editing for chromosomal modifications

  • Recombineering for markerless mutations in the chromosome

  Functional evaluation framework:

  • Growth phenotyping under different stress conditions (especially acid stress)

  • ATPase activity measurements compared to wild-type (as in the documented mutations that reduced activity by 5.61-43.44%)

  • Proton pumping efficiency

  • Protein stability and complex assembly analysis

  Data analysis approach:

  • Structure-based interpretation of mutation effects

  • Correlation of biochemical parameters with growth phenotypes

  • Molecular dynamics simulations to rationalize experimental findings

  • Statistical analysis to ensure reproducibility and significance

  This systematic approach would allow researchers to build a comprehensive understanding of structure-function relationships in L. plantarum atpF.

Application Questions

• How can recombinant L. plantarum ATP synthase be utilized to study bacterial adaptation to acid stress?

  Recombinant L. plantarum ATP synthase provides an excellent model system for studying bacterial acid stress adaptation mechanisms:

  Research design approach:

  • Generate a panel of recombinant L. plantarum strains with mutations in atpF and other ATP synthase subunits

  • Create strains with fluorescently tagged ATP synthase subunits for localization studies

  • Develop reporter strains with ATP synthase activity-linked fluorescent readouts

  Experimental methodologies:

  • Acid tolerance response (ATR) assays comparing wild-type and mutant strains

  • Real-time monitoring of intracellular pH during acid challenge

  • ATP synthesis/hydrolysis measurements at different pH values

  • Membrane integrity assessment during acid stress

  Advanced applications:

  • Correlate ATP synthase activity with expression of other acid resistance systems

  • Map the temporal sequence of acid adaptation mechanisms

  • Identify metabolic network adjustments that compensate for ATP synthase dysfunction

  • Compare acid adaptation strategies across different Lactobacillus species

  Data integration framework:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Develop computational models of acid stress response

  • Identify therapeutic targets for acid-related disorders

  This research could provide insights applicable to food fermentation, probiotic function in the gut, and bacterial pathogenesis mechanisms .

• What methodological approaches allow researchers to investigate the role of atpF in L. plantarum membrane dynamics during environmental stress?

  Investigating atpF's role in membrane dynamics during stress requires integrating multiple methodological approaches:

  Membrane composition analysis:

  • Lipidomics to quantify changes in phospholipid profiles in response to atpF mutations

  • Fatty acid methyl ester (FAME) analysis to detect changes in fatty acid composition

  • Fluorescence anisotropy measurements to assess membrane fluidity

  • Differential scanning calorimetry to determine phase transition temperatures

  Membrane protein organization studies:

  • Super-resolution microscopy (STORM/PALM) to visualize ATP synthase distribution

  • Fluorescence recovery after photobleaching (FRAP) to measure protein mobility

  • Single-particle tracking to monitor ATP synthase dynamics

  • Atomic force microscopy to map surface topography and mechanical properties

  Stress response integration:

  • Time-course analysis of membrane adaptation to different stressors

  • Correlation between ATP synthase activity and membrane physical properties

  • Assessment of ion leakage during stress exposure

  • Measurement of proton motive force maintenance during stress

  Engineering approaches:

  • Construction of chimeric atpF proteins with domains from stress-resistant organisms

  • Site-directed mutagenesis targeting membrane-interacting residues

  • Expression of modified atpF under stress-responsive promoters

  This research would help understand how L. plantarum modifies its membrane, including through de novo fatty acid biosynthesis and modification of existing lipid membrane phospholipid acyl chains, to maintain ATP synthase function under stress conditions .

• How can researchers utilize recombinant L. plantarum expressing modified ATP synthase to study bioenergetics in probiotic applications?

  Recombinant L. plantarum with modified ATP synthase offers a powerful platform for studying probiotic bioenergetics:

  Experimental design strategies:

  • Create a library of L. plantarum strains with varying ATP synthase efficiency

  • Develop dual reporter systems linking ATP production to one fluorophore and pH to another

  • Establish in vitro gut models with controlled environmental parameters

  • Design trackable L. plantarum strains for in vivo colonization studies

  Measurement approaches:

  • Real-time ATP monitoring using luciferase-based reporters

  • Assessment of metabolic outputs (organic acids, exopolysaccharides) in relation to ATP synthase activity

  • Measurement of redox balance (NAD+/NADH ratio) during gastrointestinal transit

  • Correlation between ATP synthase activity and stress resistance phenotypes

  Application methodologies:

  • Gastrointestinal survival assays comparing strains with different ATP synthase variants

  • Competition experiments between wild-type and engineered strains

  • Host-microbe interaction studies measuring immune response to different variants

  • Metabolic interaction studies with other microbiome members

  Translational research approaches:

  • Development of strain selection criteria for improved probiotic performance

  • Design of prebiotic supplements that enhance ATP production in L. plantarum

  • Identification of biomarkers for optimal bioenergetic function in probiotics

  This research direction could lead to the development of next-generation probiotics with enhanced survival in the gastrointestinal tract and improved metabolic capabilities .

Structural and Expression Questions

• What is the current understanding of proton translocation pathways through the ATP synthase complex in L. plantarum, and how can they be experimentally mapped?

  The proton translocation pathway through L. plantarum ATP synthase follows principles similar to those observed in other bacterial species, but with species-specific adaptations:

  Current structural understanding:
  Based on homology with the Bacillus PS3 ATP synthase, proton translocation likely occurs through:

  • A periplasmic half-channel that directs protons to the c-ring

  • Protonation of glutamate residues in c-subunits, causing rotation

  • A cytoplasmic half-channel where protons are released into the cytoplasm, facilitated by interaction with a conserved arginine residue (homologous to Arg169 in Bacillus PS3)

  Experimental mapping approaches:

  • Cysteine scanning mutagenesis followed by silver ion (Ag+) accessibility testing, similar to methods used for E. coli ATP synthase

  • Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy

  • Molecular dynamics simulations with explicit water molecules and ions

  • Proton transfer kinetics using pH-jump experiments and time-resolved spectroscopy

  Functional validation methods:

  • Measurement of proton/ATP ratios in reconstituted systems

  • pH-dependent activity profiling of wild-type and mutant enzymes

  • Ion selectivity studies examining competition between H+ and other ions

  • Electrophysiological recordings from membrane patches or reconstituted systems

  Integration with structural data:

  • Cryo-EM structures in different rotational states to capture the complete translocation cycle

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions involved in proton transfer

  • Computational electrostatics analysis to map potential proton pathways

  This integrated approach would provide a comprehensive understanding of the structural basis for ATP synthase function in L. plantarum and could reveal adaptations that enable function in acidic environments .

• What strategies can improve the stability and yield of recombinant L. plantarum ATP synthase subunit b for structural studies?

  Optimizing stability and yield of recombinant L. plantarum atpF requires addressing the challenges inherent to membrane protein expression and purification:

  Expression optimization:

  • Fusion protein approaches: MBP, SUMO, or Mistic fusions to improve folding and solubility

  • Directed evolution of expression hosts to select for variants that better accommodate membrane protein expression

  • Co-expression with chaperones (GroEL/ES, DnaK/J) to assist proper folding

  • Testing of multiple L. plantarum atpF variants with terminal truncations to identify more stable constructs

  Solubilization strategies:

  • Detergent screening: Systematic testing of different detergent classes (maltoside, glucoside, fos-choline)

  • Lipid supplementation during solubilization to maintain native-like environment

  • Use of styrene-maleic acid copolymer (SMA) for native nanodiscs formation

  • Amphipol substitution for long-term stability

  Purification optimization:

  • Temperature control throughout the purification process

  • Addition of stabilizing ligands or lipids in purification buffers

  • Implementation of high-throughput purification screening to identify optimal conditions

  • Use of automated systems to minimize handling time

  Stability assessment methods:

  • Thermal shift assays in different buffer conditions

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to monitor oligomeric state

  • Mass photometry to assess sample homogeneity

  • Negative stain electron microscopy for quality control

  Storage considerations:

  • Flash-freezing in liquid nitrogen with cryoprotectants

  • Lyophilization in stabilizing buffer containing trehalose (6%)

  • Storage as concentrated aliquots at -80°C

  • Avoidance of repeated freeze-thaw cycles

  These approaches should be systematically tested and optimized for L. plantarum atpF specifically, as membrane protein behavior can be highly individual.

• How do post-translational modifications of ATP synthase subunits in L. plantarum affect enzyme function, and what methods are best for characterizing them?

  Post-translational modifications (PTMs) of ATP synthase subunits can significantly impact enzyme function, though they remain less characterized in L. plantarum compared to other organisms:

  Potential PTMs of interest:

  • Phosphorylation: May regulate ATP synthase activity in response to energy status

  • Acetylation: Could affect protein-protein interactions within the complex

  • Lipid modifications: Might enhance membrane association

  • Oxidative modifications: May occur during oxidative stress

  Detection methodologies:

  • Mass spectrometry-based proteomics: Bottom-up and top-down approaches

  • PTM-specific antibodies for Western blotting

  • 2D gel electrophoresis to separate modified protein variants

  • Radioactive labeling with 32P for phosphorylation studies

  Functional characterization approaches:

  • Site-directed mutagenesis of modified residues to mimic or prevent modification

  • In vitro modification systems to generate homogeneously modified protein

  • Activity comparisons between native and recombinant (potentially differently modified) proteins

  • Time-resolved studies to correlate modifications with environmental changes

  Integration with structural biology:

  • Cryo-EM analysis of ATP synthase with and without specific modifications

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes induced by PTMs

  • Molecular dynamics simulations to predict the impact of modifications on protein dynamics

  Physiological relevance investigation:

  • Correlation of PTM patterns with growth conditions and stress responses

  • Studies in PTM-deficient strains (enzyme knockout backgrounds)

  • In vivo labeling approaches to monitor modification dynamics

  This comprehensive approach would help establish how L. plantarum uses PTMs to regulate ATP synthase function in response to environmental conditions, potentially revealing unique adaptations compared to other bacterial species.