Recombinant Pseudomonas syringae pv. tomato ATP synthase subunit a (atpB)

What is the fundamental role of ATP synthase subunit a (atpB) in Pseudomonas syringae pv. tomato?

ATP synthase subunit a (atpB) in Pseudomonas syringae pv. tomato is a critical component of the F0 portion of ATP synthase, functioning as part of the membrane-embedded proton channel. This subunit facilitates proton translocation across the bacterial membrane, which drives the rotational movement of the F0 complex. This movement is coupled to conformational changes in the F1 complex, ultimately leading to ATP synthesis. Unlike eukaryotic ATP synthase located in mitochondria, the bacterial ATP synthase including the atpB subunit is embedded in the plasma membrane . The protein plays an essential role in energy metabolism, as it contributes to the primary mechanism by which the bacteria generate ATP through oxidative phosphorylation.

How does atpB structure compare between Pseudomonas syringae pv. tomato and other bacterial species?

The atpB subunit in Pseudomonas syringae pv. tomato shares structural similarities with other bacterial species, particularly within the Proteobacteria phylum. To analyze structural conservation, researchers typically employ multiple sequence alignment tools such as CLUSTAL W or MUSCLE, followed by phylogenetic analysis using maximum likelihood methods.

The structural comparison reveals that atpB typically contains:

• Multiple transmembrane alpha-helical domains that span the bacterial membrane

• Conserved arginine residues that participate in proton translocation

• Species-specific variations in non-conserved regions that may relate to environmental adaptations

This comparative analysis is essential for understanding functional variations across bacterial species and can inform experimental design for site-directed mutagenesis studies targeting conserved functional domains.

What are the known post-translational modifications of atpB in Pseudomonas species?

Post-translational modifications (PTMs) of atpB in Pseudomonas species include phosphorylation, acetylation, and in some cases, glycosylation. These modifications can be studied using:

• Mass spectrometry-based proteomics approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • MALDI-TOF MS analysis of tryptic digests

• Site-specific antibodies that recognize modified residues

• Phosphoproteomic analysis using:

  • Immobilized metal affinity chromatography (IMAC)

  • Titanium dioxide (TiO2) enrichment followed by LC-MS/MS

These PTMs play critical roles in regulating ATP synthase activity, particularly in response to environmental stressors such as pH changes, nutrient limitation, or oxidative stress. Phosphorylation events, especially on serine and threonine residues, have been implicated in modulating proton conductance through the F0 complex.

What are the optimal conditions for heterologous expression of recombinant P. syringae pv. tomato atpB?

The optimal conditions for heterologous expression of recombinant P. syringae pv. tomato atpB require careful optimization of expression systems, growth conditions, and induction parameters:

Expression Systems:

• Escherichia coli expression systems:

  • BL21(DE3) strain with pET vector systems (pET28a or pET22b)

  • C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression

  • ArcticExpress strains for low-temperature expression to improve protein folding

• Expression parameters:

  • Induction at OD600 = 0.6-0.8

  • IPTG concentration: 0.1-0.5 mM

  • Post-induction temperature: 16-25°C (lower temperatures often yield better results for membrane proteins)

  • Expression duration: 16-20 hours

Culture Media Optimization:

For optimal results, expression trials should test multiple combinations of strains, vectors, media, and induction conditions, with expression levels monitored by Western blot using anti-His or custom anti-atpB antibodies .

What purification strategy yields the highest purity and activity for recombinant atpB protein?

A multi-step purification strategy is necessary to obtain high-purity, active recombinant atpB protein:

• Membrane isolation:

  • Harvest cells and disrupt using French press (15,000 psi, 2-3 passes) or sonication

  • Remove unbroken cells and debris by centrifugation (10,000 × g, 20 min)

  • Isolate membranes by ultracentrifugation (150,000 × g, 1 hour)

• Solubilization:

  • Solubilize membrane proteins using detergents such as:

    • n-Dodecyl β-D-maltoside (DDM): 1-2% (w/v)

    • Digitonin: 1-2% (w/v)

    • CHAPS: 1% (w/v)

  • Solubilize for 1-2 hours at 4°C with gentle rotation

• Affinity chromatography:

  • For His-tagged constructs: Ni-NTA or TALON resin

  • Wash with 20-50 mM imidazole to reduce non-specific binding

  • Elute with 250-500 mM imidazole gradient

• Size exclusion chromatography:

  • Superdex 200 column equilibrated with buffer containing 0.05-0.1% detergent

  • Flow rate: 0.5 ml/min

• Quality control assessments:

  • SDS-PAGE analysis (>95% purity)

  • Western blot confirmation

  • Mass spectrometry verification

Maintaining detergent concentration above critical micelle concentration (CMC) throughout all purification steps is crucial for preserving protein structure and activity . The use of stabilizing agents such as glycerol (10%) and reducing agents like DTT (1 mM) in all buffers helps maintain protein integrity.

How can researchers overcome challenges in expressing functional recombinant membrane proteins like atpB?

Expressing functional recombinant membrane proteins like atpB presents several challenges that can be overcome using specialized approaches:

• Toxicity issues:

  • Use tightly regulated expression systems (e.g., pBAD vectors)

  • Employ specialized E. coli strains like C41(DE3) and C43(DE3) designed for toxic membrane proteins

  • Utilize lower-copy-number vectors to reduce basal expression

• Protein misfolding:

  • Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Include chemical chaperones in growth media (e.g., 4% ethanol, 0.5 M sorbitol)

  • Optimize growth temperature (typically 16-25°C)

• Inclusion body formation:

  • If unavoidable, develop refolding protocols using:

    • Step-wise dialysis with decreasing concentrations of chaotropic agents

    • Pulse refolding method

    • Use of lipid/detergent mixed micelles during refolding

• Low yield:

  • Scale-up cultivation using bioreactors with controlled parameters

  • Optimize codon usage for E. coli expression

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

• Alternative expression systems:

  • Cell-free protein synthesis systems supplemented with lipids/detergents

  • Pseudomonas-based expression systems for homologous expression

  • Bacillus subtilis expression for Gram-positive bacterial environments

These strategies have been successfully applied to other membrane proteins in Pseudomonas species, as demonstrated by subcellular fractionation and localization studies of membrane-associated proteins . The key is to maintain the native membrane environment during extraction and purification to preserve functional activity.

What are the most effective methods for determining the structure of recombinant atpB protein?

Determining the structure of recombinant atpB protein requires specialized approaches for membrane proteins:

• X-ray crystallography:

  • Lipidic cubic phase (LCP) or bicelle crystallization

  • Vapor diffusion with detergent-solubilized protein

  • Antibody fragment co-crystallization to stabilize flexible regions

  • Typical resolution achieved: 2.5-3.5 Å

• Cryo-electron microscopy (Cryo-EM):

  • Single-particle analysis using 300 kV electron microscopes

  • Processing with software packages like RELION or cryoSPARC

  • Advantages: no need for crystals, captures multiple conformational states

  • Typical resolution for membrane proteins: 3-4 Å

• Nuclear Magnetic Resonance (NMR):

  • Solution NMR for smaller domains

  • Solid-state NMR for full-length protein in lipid environments

  • Selective isotope labeling (13C, 15N, 2H) for specific residue analysis

  • Best for dynamics studies and ligand binding

• Integrative structural biology approaches:

  • Combine low-resolution techniques (SAXS, SANS) with high-resolution methods

  • Molecular dynamics simulations to model protein-lipid interactions

  • Crosslinking mass spectrometry to identify spatial relationships

• Computational prediction methods:

  • AlphaFold2 or RoseTTAFold for initial structural models

  • Molecular dynamics refinement in explicit membrane environments

  • Rigorous validation using experimental constraints

The structural analysis of atpB is critical for understanding its functional mechanism within the ATP synthase complex and can reveal potential sites for mutagenesis studies targeting the proton channel functionality .

How can researchers accurately measure the ATPase activity of recombinant atpB in vitro?

Accurately measuring ATPase activity of recombinant atpB requires specialized assays that account for its role within the ATP synthase complex:

• Reconstitution into proteoliposomes:

  • Mix purified atpB with lipid mixtures (POPC/POPE/cardiolipin, 70:20:10)

  • Remove detergent using Bio-Beads or dialysis

  • Verify incorporation by freeze-fracture electron microscopy

  • Establish proton gradient using acid-base transition or valinomycin/K+

• Coupled enzyme assays:

  • ATP hydrolysis coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Continuous spectrophotometric monitoring at 340 nm

  • Calculate activity using extinction coefficient of NADH (6,220 M-1 cm-1)

• Phosphate release assays:

  • Malachite green assay for endpoint measurements

  • EnzChek Phosphate Assay Kit for continuous monitoring

  • Standard curve ranging from 0.1-10 μM phosphate

• Luciferase-based ATP detection:

  • Highly sensitive for measuring ATP synthesis

  • Linear detection range: 10-9 to 10-6 M ATP

  • Requires careful control of background ATP levels

Typical activity parameters for wildtype atpB:

These assays should include appropriate controls, such as known ATPase inhibitors (oligomycin, DCCD) and uncouplers (CCCP) to validate the specific activity of properly assembled ATP synthase complexes containing atpB .

What techniques are most reliable for studying atpB protein-protein interactions within the ATP synthase complex?

Studying atpB protein-protein interactions within the ATP synthase complex requires techniques that preserve native contacts:

• Chemical cross-linking coupled with mass spectrometry (XL-MS):

  • Cross-linkers: DSS (amine-reactive), EDC (zero-length), photo-reactive compounds

  • Sample digestion and cross-linked peptide enrichment

  • LC-MS/MS analysis with specialized software (pLink, StavroX, XlinkX)

  • Provides spatial constraints for interacting residues

• Co-immunoprecipitation with subunit-specific antibodies:

  • Generate antibodies against atpB or other ATP synthase subunits

  • Perform IP under mild solubilization conditions

  • Identify interacting partners by Western blot or mass spectrometry

  • Quantify interaction strength under varying conditions

• Förster Resonance Energy Transfer (FRET):

  • Label atpB and potential partners with compatible fluorophores

  • Measure energy transfer as evidence of proximity (<10 nm)

  • Can be performed in vivo using fluorescent protein fusions

  • Allows for dynamic interaction studies

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpB on sensor chip

  • Flow potential interacting partners and measure binding kinetics

  • Determine association/dissociation constants (ka, kd, KD)

  • Requires careful control of detergent conditions

• Bacterial two-hybrid systems:

  • BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system

  • Modified yeast two-hybrid adapted for bacterial membrane proteins

  • Allows screening for interactions in near-native conditions

• Native gel electrophoresis:

  • Blue native PAGE preserves protein complexes

  • Clear native PAGE for activity staining

  • 2D native/SDS-PAGE to identify complex components

These techniques have been successfully applied to study protein-protein interactions in other bacterial systems, including the ArtR protein in P. aeruginosa, which was localized in cellular fractions using similar approaches . Combinatorial use of multiple techniques provides the most comprehensive understanding of the interaction network.

How can gene knockout or conditional expression systems be used to study atpB function in P. syringae pv. tomato?

Gene knockout or conditional expression systems provide powerful tools for studying atpB function in P. syringae pv. tomato:

• Gene knockout strategies:

  • Homologous recombination-based methods:

    • sacB-based counter-selection strategy using suicide vectors like pEX18Tc

    • Double crossover events confirmed by PCR verification and sequencing

    • Complementation assays to confirm phenotype specificity

  • CRISPR-Cas9 genome editing:

    • Design sgRNAs targeting atpB coding sequence

    • Introduce Cas9 and sgRNA via broad-host-range vectors

    • Screen for editing events by phenotypic selection and sequencing

  • Transposon mutagenesis:

    • Random insertion library followed by phenotypic screening

    • Map insertion sites by arbitrary PCR and sequencing

    • Large-scale screening for conditional lethal phenotypes

• Conditional expression systems:

  • Inducible promoter systems:

    • pBAD (arabinose-inducible)

    • pTet (tetracycline-responsive)

    • pLac (IPTG-inducible)

    • Titrate inducer concentration to achieve varied expression levels

  • Temperature-sensitive alleles:

    • Site-directed mutagenesis to generate conditional mutants

    • Characterize growth and ATP synthase activity at permissive vs. restrictive temperatures

  • Degron-based systems:

    • Fusion of atpB to destabilizing domains controlled by small molecules

    • Rapid protein depletion upon addition of inducer

• Phenotypic characterization:

  • Growth rate measurement in different carbon sources and environmental conditions

  • Membrane potential assessment using fluorescent dyes (DiSC3)

  • Intracellular ATP levels using luciferase-based assays

  • Bacterial motility and biofilm formation assays

  • Plant infection assays to assess virulence effects

Since ATP synthase is likely essential, conditional systems may be necessary to study atpB function, similar to approaches used for other essential genes in Pseudomonas species . Complementation with wild-type or mutant versions can confirm specificity of phenotypes and allow structure-function analyses.

What approaches are most effective for analyzing the contribution of atpB to bacterial fitness and virulence?

Analyzing the contribution of atpB to bacterial fitness and virulence requires multifaceted approaches:

• In vitro fitness assessments:

  • Growth curve analysis:

    • Measure growth parameters (lag time, doubling time, maximum OD)

    • Test multiple media conditions (minimal vs. rich, carbon sources)

    • Growth under stress conditions (pH, temperature, oxidative stress)

  • Competition assays:

    • Co-culture wild-type and atpB-modified strains

    • Differentiate strains using antibiotic markers or fluorescent proteins

    • Calculate competitive index (CI) after several generations

• Stress response characterization:

  • pH tolerance:

    • Growth in buffered media at pH range 5.0-8.0

    • Internal pH measurement using ratiometric dyes

  • Energy stress response:

    • Transcriptomic analysis under ATP-limiting conditions

    • Metabolomic profiling to detect compensatory pathways

    • Proteome changes using quantitative mass spectrometry

• Virulence-related phenotypes:

  • Plant infection models:

    • Tomato leaf infiltration assays

    • Disease symptom scoring and bacterial population measurements

    • In planta competitive index determination

  • Hypersensitive response:

    • Non-host plant infiltration assays

    • Cell death quantification using ion leakage measurements

  • Type III secretion system activity:

    • Western blot analysis of effector protein secretion

    • Transcriptional reporter assays for T3SS gene expression

    • Similar to approaches used for ArtR in P. aeruginosa

• Biofilm formation:

  • Static microtiter plate assays with crystal violet staining

  • Flow cell microscopy for architectural analysis

  • Extracellular polymeric substance quantification

• Advanced in vivo tracking:

  • Bioluminescent imaging to track infection in real-time

  • Confocal microscopy with fluorescently labeled strains

  • Spatial transcriptomics to map gene expression during infection

These approaches can reveal how atpB-mediated energy generation impacts various aspects of bacterial physiology and virulence, similar to how essential gene studies have been conducted in P. aeruginosa .

How do mutations in conserved residues of atpB affect proton translocation and ATP synthesis?

Mutations in conserved residues of atpB have significant effects on proton translocation and ATP synthesis, which can be assessed through carefully designed experiments:

• Site-directed mutagenesis strategies:

  • Target conserved residues identified through multiple sequence alignment

  • Focus on arginine residues in the proposed proton channel

  • Create alanine substitutions for size-neutral effects

  • Implement charge-reversal mutations (positive to negative)

  • Generate conservative substitutions (e.g., Arg to Lys) to assess charge importance

• Functional analysis of mutants:

  • ATP synthesis assays:

    • Measure ATP production in inverted membrane vesicles

    • Assess P/O ratio (ATP formed per oxygen consumed)

    • Determine maximum ATP synthesis rate and proton requirements

  • Proton translocation measurements:

    • ACMA fluorescence quenching assays

    • Radioisotope (³H⁺) uptake experiments

    • Patch-clamp electrophysiology of reconstituted proteins

• Effects on specific parameters:

• Thermodynamic analysis:

  • Measure ΔpH and ΔΨ components of proton motive force

  • Calculate thermodynamic efficiency (ATP formed/theoretical maximum)

  • Determine threshold PMF required for ATP synthesis

• Advanced biophysical characterization:

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • Molecular dynamics simulations to visualize proton paths

  • Single-molecule FRET to observe conformational dynamics

These studies can reveal the critical residues involved in coupling proton movement to ATP synthesis, similar to approaches used in other energy-coupling membrane proteins in various bacterial species .

How can high-throughput approaches be used to screen for inhibitors or modulators of atpB function?

High-throughput screening approaches for identifying inhibitors or modulators of atpB function can be implemented using the following methodologies:

• In vitro ATP synthase activity assays:

  • Miniaturized coupled enzyme assays:

    • Adapt standard ATPase assays to 384 or 1536-well formats

    • Measure NADH absorbance decrease at 340 nm

    • Z' factor optimization for robust screening (target Z' > 0.7)

  • Bioluminescence-based ATP detection:

    • Use luciferase reaction to quantify ATP synthesis

    • Amenable to ultra-high-throughput formats

    • Higher sensitivity than coupled enzyme assays

• Whole-cell based screens:

  • Growth inhibition assays:

    • Wild-type vs. atpB-sensitized strains

    • Differential sensitivity indicates target specificity

    • Calculate selectivity index for each compound

  • Reporter-based systems:

    • GFP fusion to monitor atpB expression/stability

    • Stress-responsive promoters to detect ATP synthase inhibition

    • Membrane potential-sensitive fluorescent dyes (DiSC3, JC-1)

• Fragment-based screening approaches:

  • Surface plasmon resonance with immobilized atpB

  • Thermal shift assays to detect binding-induced stabilization

  • NMR-based fragment screening for binding site identification

• In silico screening methods:

  • Structure-based virtual screening against atpB binding sites

  • Molecular docking of compound libraries

  • Pharmacophore modeling based on known ATP synthase inhibitors

  • Molecular dynamics simulations to identify allosteric sites

• Screening data analysis and validation:

• Target validation techniques:

  • Generation of resistant mutants

  • Overexpression of atpB to verify target

  • Direct binding measurements (SPR, ITC)

  • Chemical proteomics to confirm in-cell target engagement

These approaches can be used to discover compounds that specifically target atpB function, potentially leading to new antibacterial agents or chemical biology tools to study ATP synthase function, similar to approaches used to identify vulnerabilities in P. aeruginosa metabolism .

What are the cutting-edge approaches for studying the dynamics of atpB within the ATP synthase complex?

Cutting-edge approaches for studying atpB dynamics within the ATP synthase complex include:

• Time-resolved structural methods:

  • Time-resolved cryo-EM:

    • Capture distinct conformational states during catalytic cycle

    • Mixing-spraying techniques for millisecond time resolution

    • 3D classification to identify conformational ensembles

  • Time-resolved X-ray free electron laser (XFEL) crystallography:

    • Pump-probe experiments with sub-picosecond resolution

    • Capture transient intermediate states during proton translocation

    • Microcrystal delivery systems for serial femtosecond crystallography

• Advanced spectroscopic techniques:

  • Single-molecule FRET:

    • Monitor distance changes between labeled subunits in real-time

    • Track rotational movements of c-ring relative to atpB

    • Analyze dwell times and step sizes during ATP synthesis

  • Electron paramagnetic resonance (EPR) spectroscopy:

    • Site-directed spin labeling of specific atpB residues

    • Distance measurements between spin labels (DEER/PELDOR)

    • Local environment changes during catalytic cycle

• High-speed atomic force microscopy (HS-AFM):

  • Visualize conformational changes at nanometer resolution

  • Capture ATP synthase rotary motion in native-like lipid environments

  • Correlate structural changes with biochemical states

• Advanced computational approaches:

  • Molecular dynamics simulations:

    • Enhanced sampling methods (metadynamics, umbrella sampling)

    • Coarse-grained simulations for longer timescales

    • Computational electrophysiology to model proton translocation

  • Quantum mechanics/molecular mechanics (QM/MM):

    • Model proton transfer events with quantum accuracy

    • Calculate energy barriers for critical steps

    • Predict effects of mutations on proton pathway

• In-cell structural biology:

  • Cryo-electron tomography:

    • Visualize ATP synthase in its native cellular context

    • Subtomogram averaging for higher resolution

    • Correlative light and electron microscopy for specific targeting

  • In-cell NMR:

    • Isotope labeling of atpB in living bacteria

    • Monitor structural changes in response to metabolic shifts

    • Detect ligand binding events in the native environment

These cutting-edge approaches can provide unprecedented insights into how atpB facilitates proton movement and contributes to the rotary mechanism of ATP synthase, building upon knowledge of energy-coupling membrane proteins described in the literature .

How can systems biology approaches integrate atpB function into broader metabolic and virulence networks?

Systems biology approaches can effectively integrate atpB function into broader metabolic and virulence networks through multi-omics and computational modeling:

• Multi-omics integration:

  • Transcriptomics:

    • RNA-seq comparison of wild-type vs. atpB-modulated strains

    • Identification of compensatory expression networks

    • Time-course analysis during environmental transitions

  • Proteomics:

    • Global proteome changes using quantitative mass spectrometry

    • Phosphoproteomics to identify regulatory networks

    • Protein-protein interaction networks via proximity labeling (BioID, APEX)

  • Metabolomics:

    • Central carbon metabolite profiling

    • Energy charge ratio (ATP/ADP/AMP) measurements

    • Flux analysis using 13C-labeled substrates

  • Multi-omics data integration:

    • Correlation networks across datasets

    • Pathway enrichment analysis

    • Causal network inference algorithms

• Genome-scale metabolic modeling:

  • Constraint-based modeling (flux balance analysis)

  • Integration of transcriptomic data for condition-specific models

  • Prediction of metabolic vulnerabilities upon atpB perturbation

  • Similar to essential gene function assessment in P. aeruginosa

• Network analysis approaches:

  • Protein-protein interaction networks centered on ATP synthase

  • Regulatory network mapping through ChIP-seq of transcription factors

  • Epistasis mapping through double-mutant phenotypic analysis

  • Network perturbation analysis to identify key control points

• Advanced computational modeling:

• Experimental validation of systems predictions:

  • Targeted gene knockouts of predicted interaction partners

  • Chemical genetic approaches with metabolic inhibitors

  • Synthetic lethality screens to validate network connections

  • Similar to methodology used for identifying condition-specific essential genes

These systems biology approaches can reveal how atpB and ATP synthase function is integrated with other cellular processes, providing a holistic understanding of bacterial physiology and identifying potential intervention points for antimicrobial development.